Signal boost cascade assay

ABSTRACT

The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods provide signal boost upon detection of target nucleic acids of interest in less than one minute and in some instances instantaneously at ambient temperatures down to 16° C. or less, without amplification of the target nucleic acids yet allowing for massive multiplexing, high accuracy and minimal non-specific signal generation.

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 63/289,112, filed 13 Dec. 2021; U.S. Ser. No. 63/359,183, filed 7 Jul. 2022; U.S. Ser. No. 63/395,394, filed 5 Aug. 2022; and U.S. Ser. No. 63/397,785, filed 12 Aug. 2022.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

Submitted herewith is an electronically filed sequence listing via EFS-Web a Sequence Listing XML, entitled “LS004US1_seglist_20221201”, created 1 Dec. 2022, which is 1,227,000 bytes in size. The sequence listing is part of the specification of this specification and is incorporated by reference in its entirety.

PETITION UNDER 37 CFR 1.84(a)(2)

This patent application contains at least one drawing executed in color. The color drawings are necessary as the only practical medium by which aspects of the claimed subject matter may be accurately conveyed. The claimed invention relates to variant proteins that alter the active site thereof and the color drawings are necessary to easily discern the structural difference between variants. As the color drawings are being filed electronically via EFS-Web, only one set of the drawings is required.

FIELD OF THE INVENTION

The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods provide a signal boost upon detection of target nucleic acids of interest in less than one minute and at ambient temperatures down to 16° C. or less.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.

Rapid and accurate identification of, e.g., infectious agents, microbe contamination, variant nucleic acid sequences that indicate the present of diseases such as cancer or contamination by heterologous sources is important in order to select correct treatment; identify tainted food, pharmaceuticals, cosmetics and other commercial goods; and to monitor the environment including identification of biothreats. Classic PCR and nucleic acid-guided nuclease or CRISPR (clustered regularly interspaced short palindromic repeats) detection methods rely on pre-amplification of target nucleic acids of interest to enhance detection sensitivity. However, amplification increases time to detection and may cause changes to the relative proportion of nucleic acids in samples that, in turn, lead to artifacts or inaccurate results. Improved technologies that allow very rapid and accurate detection of nucleic acids are therefore needed for timely diagnosis and treatment of disease, to identify toxins in consumables and the environment, as well as in other applications.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.

The present disclosure provides compositions of matter and assay methods to detect target nucleic acids of interest. The “nucleic acid-guided nuclease cascade assays” or “signal boost cascade assays” or “cascade assays” described herein comprise two different ribonucleoprotein complexes and either blocked nucleic acid molecules or blocked primer molecules. The blocked nucleic acid molecules or blocked primer molecules keep one of the ribonucleoprotein complexes “locked” unless and until a target nucleic acid of interest activates the other ribonucleoprotein complex. The present nucleic acid-guided nuclease cascade assay can detect one or more target nucleic acids of interest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits in less than one minute and in some embodiments virtually instantaneously without the need for amplifying the target nucleic acid(s) of interest, thereby avoiding the drawbacks of multiplex DNA amplification, such as primer-dimerization. Further, the cascade assay prevents “leakiness” that can lead to non-specific signal generation resulting in false positives by preventing unwinding of the blocked nucleic acid molecules or blocked primer molecules (double-stranded molecules); thus, the cascade assay is quantitative in addition to being rapid. A particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in RNP1, the cascade assay components are the same in each assay no matter what target nucleic acid(s) of interest is being detected; moreover, the gRNA in the RNP1 is easily reprogrammed using traditional guide design methods.

The present disclosure is related first, to the instantaneous cascade assay, and second, to three modalities for preventing any “leakiness” in the cascade assay leading to false positives. The three modalities enhance the cascade assay and are in addition to using blocked nucleic acid molecules or blocked primer molecules in the cascade assay.

A first embodiment provides a method for identifying a target nucleic acid of interest in a sample in one minute or less at 16° C. or more comprising the steps of: providing a reaction mixture comprising: first ribonucleoprotein complexes (RNP1s) each comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest; and wherein binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; a plurality of the blocked nucleic acid molecules comprising a sequence corresponding to the second gRNA, wherein the blocked nucleic acid molecules comprise: a first region recognized by the RNP2 complex; one or more second regions not complementary to the first region forming at least one loop; one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the plurality of blocked nucleic acid molecules and the RNP2s optionally are at a concentration ratio where the blocked nucleic acid molecules are at an equal or higher molar concentration than the RNP2s in the reaction mixture, wherein the blocked nucleic acid molecules optionally each comprise at least one bulky modification, and wherein the reaction mixture comprises at least one of a variant nuclease, the concentration ratio of the blocked nucleic acid molecules at a higher molar concentration than the molar concentration of RNP2s in the reaction mixture, and/or the blocked nucleic acid molecules comprise at least one bulky modification; contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample to bind to RNP1, wherein upon binding of the target nucleic acid of interest RNP1 becomes active initiating trans-cleavage of at least one of the plurality of blocked nucleic acid molecules thereby producing at least one unblocked nucleic acid molecule, and wherein the at least one unblocked nucleic acid molecule binds to RNP2 initiating trans-cleavage of at least one further blocked nucleic acid molecule; and detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample in one minute or less.

An additional embodiment provides a method for identifying a target nucleic acid of interest in a sample in one minute or less at 16° C. or more comprising the steps of: providing a reaction mixture comprising: first ribonucleoprotein complexes (RNP1s), wherein the RNP1s comprise a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to the nucleic acid target of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on a synthesized activating molecule, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; a plurality of template molecules comprising sequence homology to the second gRNA; a plurality of the blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked primer molecules cannot be extended by a polymerase, and wherein the blocked primer molecules comprise: a first region recognized by the RNP2; one or more second regions not complementary to the first region forming at least one loop; and one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the plurality of blocked primer molecules and the RNP2s optionally are at a concentration ratio where the blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, wherein the blocked primer molecules each optionally comprise at least one bulky modification, and wherein the reaction mixture comprises at least one of a variant nuclease, a concentration ratio where the blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or the blocked nucleic acid molecules comprising at least one bulky modification; and a polymerase and a plurality of nucleotides; contacting the reaction mixture with the sample under conditions that allow nucleic acid targets of interest in the sample to bind to RNP1, wherein: upon binding of the nucleic acid targets of interest to the RNP1, the RNP1 becomes active trans-cleaving at least one of the blocked primer molecules, thereby producing at least one unblocked primer molecule that can be extended by the polymerase; the at least one unblocked primer molecule binds to one of the template molecules and is extended by the polymerase and nucleotides to form at least one synthesized activating molecule having a sequence complementary to the second gRNA; and the at least one synthesized activating molecule binds to the second gRNA, and RNP2 becomes active cleaving at least one further blocked primer molecule and at least one reporter moiety in a cascade; allowing the cascade to continue; and detecting the unblocked primer molecules, thereby detecting the target nucleic acid of interest in the sample in one minute or less.

Aspects of the embodiments of the methods for identifying a target nucleic acid of interest in a sample in one minute or less can be substituted for any assay for identifying target nucleic acids; for example, for detecting human pathogens; animal pathogens; disease biomarkers; pathogens in laboratories, food processing facilities, hospitals, and in the environment, including bioterrorism applications (see the exemplary organisms listed in Tables 1, 2, 3, 5 and 6 and the exemplary human biomarkers listed in Table 4). Suitable samples for testing include any environmental sample, such as air, water, soil, surface, food, clinical sites and products, industrial sites and products, pharmaceuticals, medical devices, nutraceuticals, cosmetics, personal care products, agricultural equipment and sites, and commercial samples, and any biological sample obtained from an organism or a part thereof, such as a plant, animal (including humans), or microbe.

There is also provided in an embodiment a method of detecting a target nucleic acid molecule in a sample in a cascade reaction comprising the steps of: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA) comprising a sequence complementary to a target nucleic acid molecule; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second guide RNA, (b) contacting the target nucleic acid molecule with the reaction mixture under conditions that, relative to a control reaction, reduce the probability of R-loop formation between the second gRNA and the plurality of blocked nucleic acid molecules, wherein: (i) upon binding of the target nucleic acid molecule, the RNP1 becomes active wherein the first nucleic acid-guided nuclease cleaves at least one of the blocked nucleic acid molecules, thereby producing at least one unblocked nucleic acid molecule; and (ii) at least one unblocked nucleic acid molecule binds to the second gRNA, and the RNP2 becomes active wherein the second nucleic acid-guided nuclease cleaves at least one further blocked nucleic acid molecule; and (c) detecting the cleavage products of step (b), thereby detecting the target nucleic acid molecule in the sample.

There is also provided a second embodiment comprising a method of increasing the efficiency, reducing the background, increasing the signal-to-noise ratio, reducing cis-cleavage of blocked nucleic acid molecules and preventing unwinding of the second ribonucleoprotein complex (RNP2) in a cascade reaction comprising: (a) a reaction mixture comprising: (i) a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA) comprising a sequence complementary to a target nucleic acid molecule; (ii) the RNP2 comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second guide RNA, and (b) the target nucleic acid molecule comprising a sequence complementary to the first gRNA; and the method comprising the step of initiating the cascade reaction by contacting (a) and (b) under conditions that reduce the probability of R-loop formation between the blocked nucleic acid molecules and the second gRNA, thereby reducing increasing the efficiency, reducing the background, increasing the signal-to-noise ratio, reducing cis-cleavage of blocked nucleic acid molecules and preventing unwinding of the RNP2 relative to a control reaction.

There is also provided in a third embodiment a method of increasing the signal-to-noise ratio in a cascade reaction comprising the steps of: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA) comprising a sequence complementary to a target nucleic acid molecule; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second guide RNA, (b) initiating the cascade reaction by contacting the target nucleic acid molecule with the reaction mixture under conditions that reduce the probability of R-loop formation between the second gRNA and the plurality of blocked nucleic acid molecules, thereby increasing the signal-to-noise ratio in the cascade reaction relative to a control reaction, wherein: (i) upon binding of the target nucleic acid molecule, the RNP1 becomes active cleaving at least one of the blocked nucleic acid molecules, thereby producing at least one unblocked nucleic acid molecule; and (ii) the least one unblocked nucleic acid molecule binds to the second gRNA, and the RNP2 becomes active cleaving at least one further blocked nucleic acid molecule; and (c) detecting the cleavage products of the cascade reaction in step (b); and (d) determining the signal-to-noise ratio of the cascade reactions in step (b).

A fourth embodiment provides a method of increasing the efficiency, reducing the background, increasing the signal-to-noise ratio, reducing cis-cleavage of blocked nucleic acid molecules and preventing unwinding of a second ribonucleoprotein complex (RNP2) in a cascade reaction comprising the steps of: (a) providing a reaction mixture comprising: a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA) comprising a sequence complementary to a target nucleic acid molecule; the RNP2 comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second guide RNA, (b) initiating the cascade reaction by contacting the target nucleic acid molecule with the reaction mixture under conditions that reduce the probability of R-loop formation between the second gRNA and the plurality of blocked nucleic acid molecules, thereby increasing the efficiency, reducing the background, increasing the signal-to-noise ratio, reducing cis-cleavage of blocked nucleic acid molecules and preventing unwinding of the RNP2 in the cascade reaction relative to a control reaction.

In some aspects of these embodiments, the conditions that reduce R-loop formation comprise one or more of the steps of: 1) providing a molar concentration of blocked nucleic acid molecules that exceeds the molar concentration of ribonucleoprotein complexes; 2) engineering the nucleic acid-guided nuclease used in the ribonucleoprotein complex to result in a variant nucleic acid-guided nuclease such that single stranded DNA is cleaved faster than double stranded DNA is cleaved; and/or 3) engineering the blocked nucleic acid molecules to include bulky modifications of a size of about 1 nm or less.

Another embodiment provides a method for preventing unwinding of blocked nucleic acid molecules in the presence of an RNP in a cascade reaction comprising the steps of: providing blocked nucleic acid molecules; providing ribonucleoprotein complexes comprising a nucleic acid-guided nuclease that exhibits both cis- and trans-cleavage activity upon activation and a gRNA that recognizes an unblocked nucleic acid molecule resulting from trans-cleavage of the blocked nucleic acid molecules; and providing a molar concentration of the blocked nucleic acid molecules that exceeds the molar concentration of ribonucleoprotein complexes; engineering the nucleic acid-guided nuclease used in the ribonucleoprotein complex to result in a variant nucleic acid-guided nuclease such that single stranded DNA is cleaved faster than double stranded DNA is cleaved; and/or 3) engineering the blocked nucleic acid molecules to include bulky modifications of a size of about 1 nm or less thereby preventing unwinding of the blocked nucleic acid molecules in the cascade reaction.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecules are blocked primer molecules.

In a further embodiment, there is provided a method for preventing unwinding of blocked nucleic acid molecules or blocked primer molecules in the presence of an RNP comprising the steps of: providing blocked nucleic acid molecules or blocked primer molecules; providing ribonucleoprotein complexes comprising a nucleic acid-guided nuclease that exhibits both cis- and trans-cleavage activity upon activation and a gRNA that recognizes an unblocked nucleic acid molecule or an unblocked primer molecule resulting from trans-cleavage of the blocked nucleic acid molecule or blocked primer molecule; and providing a molar concentration of blocked nucleic acid molecules that exceeds the molar concentration of ribonucleoprotein complexes; engineering the nucleic acid-guided nuclease used in the ribonucleoprotein complex to result in a variant nucleic acid-guided nuclease such that single stranded DNA is cleaved times faster than double stranded DNA is cleaved; and/or 3) engineering the blocked nucleic acid molecules to include bulky modifications of a size of about 1 nm or less.

Other embodiments provide a method for detecting target nucleic acid molecules in a sample in less than one minute without amplifying the target nucleic acid molecules; and instantaneously detecting target nucleic acid molecules in a sample without amplifying the target nucleic acid molecules.

In some aspects of the methods, the reaction mixture is provided at 16° C., and in some aspects, the reaction mixture is provided at 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., or 30° C. or higher.

Other embodiments provide reaction mixtures for identifying a target nucleic acid of interest in a sample in one minute or less comprising: first ribonucleoprotein (RNP1) complexes (RNP1s) each comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest; and wherein binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; and a plurality of the blocked nucleic acid molecules comprising a sequence corresponding to the second gRNA, wherein the blocked nucleic acid molecules comprise: a first region recognized by the RNP2 complex; one or more second regions not complementary to the first region forming at least one loop; one or more third regions complementary to and hybridized to the first region forming at least one clamp, and wherein the blocked nucleic acid molecules optionally each comprise at least one bulky modification, wherein the plurality of blocked nucleic acid molecules and the RNP2s optionally are at a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and wherein the reaction mixture comprises at least one of a variant nuclease, a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or blocked nucleic acid molecules comprising at least one bulky modification.

Also provided is a reaction mixture for identifying a target nucleic acid of interest in a sample in one minute or less comprising: first ribonucleoprotein complexes (RNP1s), wherein the RNP1s comprise a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to the nucleic acid target of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on synthesized activating molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; a plurality of template molecules comprising sequence homology to the second gRNA; a plurality of the blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked primer molecules cannot be extended by a polymerase, and wherein the blocked primer molecules comprise: a first region recognized by the RNP2; one or more second regions not complementary to the first region forming at least one loop; and one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the blocked primer molecules optionally each comprise at least one bulky modification and wherein the plurality of blocked primer molecules and the RNP2s optionally are at a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and wherein the reaction mixture comprises at least one of a variant nuclease, at a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or blocked nucleic acid molecules comprising at least one bulky modification; and a polymerase and a plurality of nucleotides.

Further provided is a composition of matter comprising: ribonucleoprotein complexes (RNPs) comprising a nucleic acid-guided nuclease and a gRNA that is not complementary to the target nucleic acid of interest; wherein the nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; and a plurality of the blocked nucleic acid molecules comprising a sequence corresponding to the gRNA, wherein the blocked nucleic acid molecules comprise: a first region recognized by the RNP complex; one or more second regions not complementary to the first region forming at least one loop; one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the blocked nucleic acid molecules each comprise at least one bulky modification, wherein the blocked nucleic acid molecules optionally each comprise at least one bulky modification, and wherein the plurality of blocked nucleic acid molecules and the RNP2s optionally are at a concentration ratio where the blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and wherein the composition comprises at least one of a variant nuclease, a concentration ratio where the blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or blocked nucleic acid molecules comprising at least one bulky modification; and a polymerase and a plurality of nucleotides.

Additionally provided is a composition of matter comprising: ribonucleoprotein complexes (RNPs) comprising a nucleic acid-guided nuclease and a gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; a plurality of template molecules comprising sequence homology to the gRNA; and a plurality of the blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked primer molecules cannot be extended by a polymerase, and wherein the blocked primer molecules comprise: a first region recognized by the RNP2; one or more second regions not complementary to the first region forming at least one loop; and one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the blocked primer molecules optionally each comprise at least one bulky modification, and wherein the plurality of blocked primer molecules and the RNPs optionally are at a concentration where the blocked nucleic acid molecules are at a molar concentration equal to or greater than the molar concentration of the RNPs in the reaction mixture, and wherein the composition comprises at least one of a variant nuclease, a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or blocked nucleic acid molecules comprising at least one bulky modification; and a polymerase and a plurality of nucleotides.

In some aspects of these embodiments, the reaction mixture further comprises reporter moieties, wherein the reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP2 to identify the presence of one or more nucleic acid targets of interest in the sample. In some aspects, the reporter moieties are not coupled to the blocked primer molecules, and wherein upon cleavage by RNP2, a signal from the reporter moiety is detected; yet in other aspects, the reporter moieties are coupled to the blocked primer molecules, and wherein upon cleavage by RNP2, a signal from the reporter moiety is detected.

In some aspects of all embodiments comprising bulky modifications, the bulky modifications are about 1 nm in size, and in some aspects, the bulky modifications are about 0.9 nm, 0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm, 0.4 nm, 0.3 nm, 0.2 nm, or 0.1 nm in size. In some aspects, the bulky modifications are about 0.9 nm, 0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm, 0.4 nm, 0.3 nm, 0.2 nm, or 0.1 nm in size. In some aspects, the blocked nucleic acid molecules include bulky modifications and wherein there are two bulky modifications with one bulky modification located on the 5′ end of the blocked nucleic acid molecule and one bulky modification located on the 3′ end of the blocked nucleic acid molecule, and where the 5′ and 3′ ends comprising the two bulky modifications are less than 11 nm from one another. In other aspects, the bulky modification is on a 5′ end of blocked nucleic acid molecules and may be selected from the group of 5′ Fam (6-fluorescein amidite); Black Hole Quencher-1-5′; biotin TEG (15 atom triethylene glycol spacer); biotin-5′; and cholesterol TEG (15 atom triethylene glycol spacer). In other aspects, the bulky modification is on a 3′ end of the blocked nucleic acid molecules and may be selected from the group of Black Hole Quencher-1-3′; biotin-3′; and TAMRA-3′ (carboxytetramethylrhodamine). In some aspects, a bulky modification is between two internal nucleic acid residues of the blocked nucleic acid molecules and may be selected from the group of Cy3 internal and Cy5, and in some aspects, the bulky modification is an internal nucleotide base modification and may be selected from the group of biotin deoxythymidine dT; disthiobiotin NHS; and fluorescein dT.

In some aspects of these embodiments, the blocked nucleic acid molecules or blocked primer molecules comprise a structure represented by any one of Formulas I-IV, wherein Formulas I-IV are in the 5′-to-3′ direction: (a)A-(B-L)J-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;     -   B is 4-12 nucleotides in length;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10;     -   C is 4-15 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then A-(B-L)J-C and T-D are separate nucleic acid         strands;     -   T is 17-135 nucleotides in length and comprises at least 50%         sequence complementarity to B and C; and     -   D is 0-10 nucleotides in length and comprises at least 50%         sequence complementarity to A;         (b)D-T-T′-C-(L-B)J-A  (Formula II);     -   wherein D is 0-10 nucleotides in length;     -   T-T′ is 17-135 nucleotides in length;     -   T′ is 1-10 nucleotides in length and does not hybridize with T;     -   C is 4-15 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   L is 3-25 nucleotides in length and does not hybridize with T;     -   B is 4-12 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   J is an integer between 1 and 10;     -   A is 0-15 nucleotides in length and comprises at least 50%         sequence complementarity to D;         (c)T-D-M-A-(B-L)J-C  (Formula III);     -   wherein T is 17-135 nucleotides in length;     -   D is 0-10 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then T-D and A-(B-L)J-C are separate nucleic acid         strands;     -   A is 0-15 nucleotides in length and comprises at least 50%         sequence complementarity to D;     -   B is 4-12 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10; and     -   C is 4-15 nucleotides in length; or         (d)T-D-M-A-L_(p)-C  (Formula IV);     -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,         or 17-25);     -   D is 0-15 nucleotides in length;     -   M is 1-25 nucleotides in length;     -   A is 0-15 nucleotides in length and comprises a sequence         complementary to D; and     -   L is 3-25 nucleotides in length;     -   p is 0 or 1;     -   C is 4-15 nucleotides in length and comprises a sequence         complementary to T.

In some aspects, (a) T of Formula I comprises at least 80% sequence complementarity to B and C; (b) D of Formula I comprises at least 80% sequence complementarity to A; (c) C of Formula II comprises at least 80% sequence complementarity to T; (d) B of Formula II comprises at least 80% sequence complementarity to T; (e) A of Formula II comprises at least 80% sequence complementarity to D; (f) A of Formula III comprises at least 80% sequence complementarity to D; (g) B of Formula III comprises at least 80% sequence complementarity to T; (h) A of Formula IV comprises at least 80% sequence complementarity to D; and/or (i) C of Formula IV comprises at least 80% sequence complementarity to T.

In some aspects, the variant nucleic acid-guided nuclease is a Type V variant nucleic acid-guided nuclease. In some aspects, the one or both of the RNP1 and the RNP2 comprise a nucleic acid-guided nuclease selected from Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, or Cas13b.

In some aspects of the embodiments that comprise a variant nucleic acid-guided nuclease, the variant nucleic acid-guided nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules wherein the mutation is selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs. In some embodiments, there are at least two mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs and in other aspects, there are at least three mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs. In some aspects, the variant nucleic acid-guided nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, wherein the at least one mutation is selected from mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:2; mutations to amino acid residues K534, Y538 and R591 in relation to SEQ ID NO:3; mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:4; mutations to amino acid residues K579, N583 and K635 in relation to SEQ ID NO:5; mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:6; mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:7; mutations to amino acid residues K617, N621 and K678 in relation to SEQ ID NO:8; mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:9; mutations to amino acid residues K569, N573 and K625 in relation to SEQ ID NO:10; mutations to amino acid residues K562, N566 and K619 in relation to SEQ ID NO:11; mutations to amino acid residues K645, N649 and K732 in relation to SEQ ID NO:12; mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:13; mutations to amino acid residues K592, N596 and K653 in relation to SEQ ID NO:14; or mutations to amino acid residues K521, N525 and K577 in relation to SEQ ID NO:15.

In some aspects, the variant nucleic acid-guided nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, wherein single stranded DNA is cleaved 1.2 to 2.5 times faster than double stranded DNA is cleaved, at least three to four times faster than double stranded DNA is cleaved, and in some aspects, single stranded DNA is cleaved at least five times faster than double stranded DNA is cleaved. In aspects, the variant nucleic acid-guided nuclease exhibits cis- and trans-cleavage activity.

In some aspects, the variant nucleic acid-guided nuclease comprises at least two mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and in some aspects, the variant nuclease comprises at least three mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules.

In any of the embodiments comprising a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, certain aspects provide that the concentration of the blocked nucleic acid molecules and the RNP2s are at a concentration ratio of at least 1.5 blocked nucleic acid molecules to 1 RNP2 in the reaction mixture, and in some aspects, the concentration of the blocked nucleic acid molecules and the RNP2s are at a concentration ratio of at least 2 blocked nucleic acid molecules to 1 RNP2 in the reaction mixture or at least 3 blocked nucleic acid molecules to 1 RNP2, or at least 3.5 blocked nucleic acid molecules to 1 RNP2, or at least 4 blocked nucleic acid molecules to 1 RNP2, or at least 4.5 blocked nucleic acid molecules to 1 RNP2, or at least 5 blocked nucleic acid molecules to 1 RNP2, or at least 5.5 blocked nucleic acid molecules to 1 RNP2, or at least 6 blocked nucleic acid molecules to 1 RNP2, or at least 6.5 blocked nucleic acid molecules to 1 RNP2, or at least 7.5 blocked nucleic acid molecules to 1 RNP2, or at least 7.5 blocked nucleic acid molecules to 1 RNP2, or at least 8 blocked nucleic acid molecules to 1 RNP2, or at least 8.5 blocked nucleic acid molecules to 1 RNP2, or at least 9 blocked nucleic acid molecules to 1 RNP2, or at least 9.5 blocked nucleic acid molecules to 1 RNP2, or at least 10 blocked nucleic acid molecules to 1 RNP2.

In further embodiments there is provided a variant Cas12a nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant Cas12a nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules and wherein the variant Cas12a nuclease exhibits both cis- and trans-cleavage activity. In some aspects, wherein the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:2; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K534, Y538 and R591 in relation to SEQ ID NO:3; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:4; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K579, N583 and K635 in relation to SEQ ID NO:5; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:6; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:7; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K617, N621 and K678 in relation to SEQ ID NO:8; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:9; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K569, N573 and K625 in relation to SEQ ID NO:10; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K562, N566 and K619 in relation to SEQ ID NO:11; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K645, N649 and K732 in relation to SEQ ID NO:12; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:13; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K592, N596 and K653 in relation to SEQ ID NO:14; or the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K521, N525 and K577 in relation to SEQ ID NO:15 including and equivalent amino acid residues in Cas12a orthologs to these SEQ ID Nos: 1-15.

In some aspects, the variant Cas12a nuclease that has been engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved comprises any one of SEQ ID NOs: 16-600.

Alternatively, an embodiment provides a single-strand-specific Cas12a nucleic acid-guided nucleases comprising an LbCas12a (i.e., SEQ ID NO: 1) with an acetylated K595 (K595K^(Ac)) residue; an AsCas12a (i.e., SEQ ID NO: 2) with an acetylated K607 (K607K^(Ac)) residue; a CtCas12a (i.e., SEQ ID NO: 3) with an acetylated R591 (R591R^(Ac)) residue; an EeCas12a (i.e., SEQ ID NO: 4) with an acetylated K601 (K607K^(Ac)) residues; an Mb3Cas12a (i.e., SEQ ID NO: 5) with an acetylated K635 (K635K^(Ac)) residue; an FnCas12a (i.e., SEQ ID NO: 6) with an acetylated K671 (K671K^(Ac)) residue; an FnoCas12a (i.e., SEQ ID NO: 7) with an acetylated N671 (N671K^(Ac)) residue; an FbCas12a (i.e., SEQ ID NO: 8) with an acetylated K678 (K678K^(Ac)) residue; an Lb4Cas12a (i.e., SEQ ID NO: 9) with an acetylated K601 (K601K^(Ac)) residue; an MbCas12a (i.e., SEQ ID NO: 10) with an acetylated K625 (K625K^(Ac)) residue; a Pb2Cas12a (i.e., SEQ ID NO: 11) with an acetylated K619 (K619K^(Ac)) residue; a PgCas12a (i.e., SEQ ID NO: 12) with an acetylated K732 (K732K^(Ac)) residue; an AaCas12a (i.e., SEQ ID NO: 13) with an acetylated K607 (K607K^(Ac)) residue; a BoCas12a (i.e., SEQ ID NO: 14) with an acetylated K653 (K653K^(Ac)) residue; or an CmaCas12a (i.e., SEQ ID NO: 15) with an acetylated K577 (K577K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a Cas12a ortholog acetylated at the amino acid of the ortholog equivalent to K595 of SEQ ID NO:1.

These aspects and other features and advantages of the invention are described below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1A is an overview of a prior art quantitative PCR (“qPCR”) assay where target nucleic acids of interest from a sample are amplified before detection.

FIG. 1B is an overview of the general principles underlying the nucleic acid-guided nuclease cascade assay described in detail herein where target nucleic acids of interest from a sample do not need to be amplified before detection.

FIG. 1C is an illustration of the unwinding issue that is mitigated by the modalities described herein.

FIG. 2A is a diagram showing the sequence of steps in an exemplary cascade assay utilizing blocked nucleic acid molecules.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule and a method for unblocking the blocked nucleic acid molecules of the disclosure.

FIG. 2C shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula I, as described herein.

FIG. 2D shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula II, as described herein.

FIG. 2E shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula III, as described herein.

FIG. 2F shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula IV, as described herein.

FIG. 2G shows an exemplary single-stranded blocked nucleic acid molecule with a design able to block R-loop formation with an RNP complex, thereby blocking activation of the trans-nuclease activity of an RNP complex (i.e., RNP2).

FIG. 2H shows schematics of exemplary circularized blocked nucleic acid molecules.

FIG. 3A is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and linear template molecules.

FIG. 3B is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and circular template molecules.

FIG. 4 illustrates three embodiments of reporter moieties.

FIG. 5 is a simplified block diagram of an exemplary method for designing, synthesizing and screening variant nucleic acid-guided nucleases.

FIG. 6A shows the result of protein structure prediction using Rosetta and SWISS modeling of wildtype LbCas12a (Lachnospiraceae bacterium Cas12a).

FIG. 6B shows the result of example mutations on the LbCas12a protein structure prediction using Rosetta and SWISS modeling of LbCas12a and indicating the PAM regions.

FIG. 7 is a simplified diagram of acetylating the K595 amino acid in the wildtype sequence of LbCas12a (K595K^(Ac)).

FIG. 8A is an illustration of a blocked nucleic acid molecule with bulky modifications, cleavage thereof, and steric hindrance at the PAM-interacting (PI) domain in a nucleic acid-guided nuclease caused by 5′ and 3′ modifications to a blocked nucleic acid molecule.

FIG. 8B illustrates five exemplary variations of blocked nucleic acid molecules with bulky modifications.

FIGS. 8C, 8D and 8E list exemplary bulky modifications for 5′, 3′, and internal positions in blocked nucleic acid molecules.

FIG. 9 is an illustration of a lateral flow assay that can be used to detect the cleavage and separation of a signal from a reporter moiety.

FIG. 10A depicts Molecule U29 and describes the properties thereof.

FIGS. 10B-10H show the results achieved for detection of 3E4 copies, 30 copies, 3 copies and 0 copies of the mecA gene of MRSA (n=3) at 25° C. with varying conentrations of Molecule U29, RNP2 and reported moiety.

FIG. 11A shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutation G532A in the wildtype sequence.

FIG. 11B shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutation K538A in the wildtype sequence.

FIG. 11C shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutation Y542A in the wildtype sequence.

FIG. 11D shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutation K595A in the wildtype sequence.

FIG. 11E shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutations G532A, K538A, Y5442A and K595A in the wildtype sequence.

FIG. 11F shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutation K595D in the wildtype sequence.

FIG. 11G shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutation K595E in the wildtype sequence.

FIG. 11H shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutations K538A, Y542A and K595D in the wildtype sequence.

FIG. 11I shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutations K538A, Y542A and K595E in the wildtype sequence.

FIGS. 12A-12G are a series of graphs showing the time for detection of dsDNA and ssDNA both with and without PAM sequences for wildtype LbaCas12a and engineered variants of LbaCas12a.

It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.

Definitions

In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.

All of the functionalities described in connection with one embodiment of the compositions and/or methods described herein are intended to be applicable to the additional embodiments of the compositions and/or methods except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.

Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “a system” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth. Additionally, it is to be understood that terms such as “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,” “outer” that may be used herein merely describe points of reference and do not necessarily limit embodiments of the present disclosure to any particular orientation or configuration. Furthermore, terms such as “first,” “second,” “third,” etc., merely identify one of a number of portions, components, steps, operations, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention. Conventional methods are used for the procedures described herein, such as those provided in the art, and demonstrated in the Examples and various general references. Unless otherwise stated, nucleic acid sequences described herein are given, when read from left to right, in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNA, as RNA, or a combination of DNA and RNA (e.g., a chimeric nucleic acid).

Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

The term “and/or” where used herein is to be taken as specific disclosure of each of the multiple specified features or components with or without another. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the term “about,” as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

As used herein, the terms “binding affinity” or “dissociation constant” or “K_(d)” refer to the tendency of a molecule to bind (covalently or non-covalently) to a different molecule. A high K_(d) (which in the context of the present disclosure refers to blocked nucleic acid molecules or blocked primer molecules binding to RNP2) indicates the presence of more unbound molecules, and a low K_(d) (which in the context of the present disclosure refers to unblocked nucleic acid molecules or unblocked primer molecules binding to RNP2) indicates the presence of more bound molecules. In the context of the present disclosure and the binding of blocked or unblocked nucleic acid molecules or blocked or unblocked primer molecules to RNP2, low K_(d) values are in a range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high K_(d) values are in the range of 100 nM-100 μM (10 mM) and thus are about 10⁵- to 10¹⁰-fold or higher as compared to low K_(d) values.

As used herein, the terms “binding domain” or “binding site” refer to a region on a protein, DNA, or RNA, to which specific molecules and/or ions (ligands) may form a covalent or non-covalent bond. By way of example, a polynucleotide sequence present on a nucleic acid molecule (e.g., a primer binding domain) may serve as a binding domain for a different nucleic acid molecule (e.g., an unblocked primer nucleic acid molecule). Characteristics of binding sites are chemical specificity, a measure of the types of ligands that will bond, and affinity, which is a measure of the strength of the chemical bond.

As used herein, the term “blocked nucleic acid molecule” refers to nucleic acid molecules that cannot bind to the first or second RNP complex to activate cis- or trans-cleavage. “Unblocked nucleic acid molecule” refers to a formerly blocked nucleic acid molecule that can bind to the second RNP complex (RNP2) to activate trans-cleavage of additional blocked nucleic acid molecules. A “blocked nucleic acid molecule” may be a “blocked primer molecule” in some embodiments of the cascade assay.

The terms “Cas RNA-guided nucleic acid-guided nuclease” or “CRISPR nuclease” or “nucleic acid-guided nuclease” refer to a CRISPR-associated protein that is an RNA-guided nucleic acid-guided nuclease suitable for assembly with a sequence-specific gRNA to form a ribonucleoprotein (RNP) complex.

As used herein, the terms “cis-cleavage”, “cis-nucleic acid-guided nuclease activity”, “cis-mediated nucleic acid-guided nuclease activity”, “cis-nuclease activity”, “cis-mediated nuclease activity”, and variations thereof refer to sequence-specific cleavage of a target nucleic acid of interest, including an unblocked nucleic acid molecule or synthesized activating molecule, by a nucleic acid-guided nuclease in an RNP complex. Cis-cleavage is a single turn-over cleavage event in that only one substrate molecule is cleaved per event.

The term “complementary” as used herein refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen-bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” or “percent homology” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10, or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3′-TCGA-5′ is 100% complementary to the nucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-ATCGAT-5′ is 100% complementary to a region of the nucleotide sequence 5′-GCTAGCTAG-3′.

As used herein, the term “contacting” refers to placement of two moieties in direct physical association, including in solid or liquid form. Contacting can occur in vitro with isolated cells (for example in a tissue culture dish or other vessel) or in samples or in vivo by administering an agent to a subject.

The term “conservative amino acid substitution” refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains comprises glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains comprises serine and threonine; a group of amino acids having amide containing side chains comprises asparagine and glutamine; a group of amino acids having aromatic side chains comprises phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains comprises lysine, arginine, and histidine; a group of amino acids having acidic side chains comprises glutamate and aspartate; and a group of amino acids having sulfur containing side chains comprises cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

A “control” is a reference standard of a known value or range of values.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1) a crRNA region or guide sequence capable of hybridizing to the target strand of a target nucleic acid of interest, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease. The crRNA region of the gRNA is a customizable component that enables specificity in every nucleic acid-guided nuclease reaction. A gRNA can include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest to hybridize with the target nucleic acid of interest and to direct sequence-specific binding of a ribonucleoprotein (RNP) complex containing the gRNA and nucleic acid-guided nuclease to the target nucleic acid. Target nucleic acids of interest may include a protospacer adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region on the target nucleic acid of interest, including on an unblocked nucleic acid molecule or synthesized activating molecule. A gRNA may contain a spacer sequence including a plurality of bases complementary to a protospacer sequence in the target nucleic acid. For example, a spacer can contain about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more bases. The gRNA spacer may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more complementary to its corresponding target nucleic acid of interest. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences. A guide RNA may be from about 20 nucleotides to about 300 nucleotides long. Guide RNAs may be produced synthetically or generated from a DNA template.

“Modified” refers to a changed state or structure of a molecule. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, a nucleic acid molecule (for example, a blocked nucleic acid molecule) may be modified by the introduction of non-natural nucleosides, nucleotides, and/or internucleoside linkages. In another embodiment, a modified protein (e.g., a modified or variant nucleic acid-guided nuclease) may refer to any polypeptide sequence alteration which is different from the wildtype.

The terms “percent sequence identity”, “percent identity”, or “sequence identity” refer to percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence following alignment by standard techniques. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, PSI-BLAST, or Megalign software. In some embodiments, the software is MUSCLE (Edgar, Nucleic Acids Res., 32(5):1792-1797 (2004)). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, in embodiments, percent sequence identity values are generated using the sequence comparison computer program BLAST (Altschul, et al., J. Mol. Biol., 215:403-410 (1990)).

As used herein, the terms “preassembled ribonucleoprotein complex”, “ribonucleoprotein complex”, “RNP complex”, or “RNP” refer to a complex containing a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to a target nucleic acid of interest, guides the RNP to the target nucleic acid of interest and hybridizes to it. The hybridized target nucleic acid-gRNA units are cleaved by the nucleic acid-guided nuclease. In the cascade assays described herein, a first ribonucleoprotein complex (RNP1) includes a first guide RNA (gRNA) specific to a target nucleic acid of interest, and a first nucleic acid-guided nuclease, such as, for example, cas12a or cas14a for a DNA target nucleic acid, or cas13a for an RNA target nucleic acid. A second ribonucleoprotein complex (RNP2) for signal amplification includes a second guide RNA specific to an unblocked nucleic acid or synthesized activating molecule, and a second nucleic acid-guided nuclease, which may be different from or the same as the first nucleic acid-guided nuclease.

As used herein, the terms “protein” and “polypeptide” are used interchangeably. Proteins may or may not be made up entirely of amino acids.

As used herein, the term “sample” refers to tissues; cells or component parts; body fluids, including but not limited to peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. “Sample” may also refer to specimens or aliquots from food; agricultural products; pharmaceuticals; cosmetics, nutraceuticals; personal care products; environmental substances such as soil, water (from both natural and treatment sites), air, or sewer samples; industrial sites and products; and chemicals and compounds. A sample further may include a homogenate, lysate or extract. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecules.

The terms “target DNA sequence”, “target sequence”, “target nucleic acid of interest”, “target molecule of interest”, “target nucleic acid”, or “target of interest” refer to any locus that is recognized by a gRNA sequence in vitro or in vivo. The “target strand” of a target nucleic acid of interest is the strand of the double-stranded target nucleic acid that is complementary to a gRNA. The spacer sequence of a gRNA may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99% or more complementary to the target nucleic acid of interest. Optimal alignment can be determined with the use of any suitable algorithm for aligning sequences. Full complementarity is not necessarily required provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of an RNP complex. A target nucleic acid of interest can include any polynucleotide, such as DNA (ssDNA or dsDNA) or RNA polynucleotides. A target nucleic acid of interest may be located in the nucleus or cytoplasm of a cell such as, for example, within an organelle of a eukaryotic cell, such as a mitochondrion or a chloroplast, or it can be exogenous to a host cell, such as a eukaryotic cell or a prokaryotic cell. The target nucleic acid of interest may be present in a sample, such as a biological or environmental sample, and it can be a viral nucleic acid molecule, a bacterial nucleic acid molecule, a fungal nucleic acid molecule, or a polynucleotide of another organism, such as a coding or a non-coding sequence, and it may include single-stranded or double-stranded DNA molecules, such as a cDNA or genomic DNA, or RNA molecules, such as mRNA, tRNA, and rRNA. The target nucleic acid of interest may be associated with a protospacer adjacent motif (PAM) sequence, which may include a 2-5 base pair sequence adjacent to the protospacer. In some embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more target nucleic acids can be detected by the disclosed method.

As used herein, the terms “trans-cleavage”, “trans-nucleic acid-guided nuclease activity”, “trans-mediated nucleic acid-guided nuclease activity”, “trans-nuclease activity”, “trans-mediated nuclease activity” and variations thereof refer to indiscriminate, non-sequence-specific cleavage of a target nucleic acid molecule by a nucleic acid-guided nuclease (such as by a Cas12, Cas13, and Cas14) which is triggered by binding of N nucleotides of a target nucleic acid molecule to a gRNA and/or by cis- (sequence-specific) cleavage of a target nucleic acid molecule. Trans-cleavage is a “multiple turn-over” event, in that more than one substrate molecule is cleaved after initiation by a single turn-over cis-cleavage event.

Type V CRISPR/Cas nucleic acid-guided nucleases are a subtype of Class 2 CRISPR/Cas effector nucleases such as, but not limited to, engineered Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), CasY (Cas12d), Cas 13a nucleases or naturally-occurring proteins, such as a Cas12a isolated from, for example, Francisella tularensis subsp. novicida (Gene ID: 60806594), Candidatus Methanoplasma termitum (Gene ID: 24818655), Candidatus Methanomethylophilus alvus (Gene ID: 15139718), and [Eubacterium] eligens ATCC 27750 (Gene ID: 41356122), and an artificial polypeptide, such as a chimeric protein.

The term “variant” in the context of the present disclosure refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many if not most regions, identical. A variant and reference polypeptide may differ in one or more amino acid residues (e.g., substitutions, additions, and/or deletions). A variant of a polypeptide may be a conservatively modified variant. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code (e.g., a non-natural amino acid). A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Variants include modifications—including chemical modifications—to one or more amino acids that do not involve amino acid substitutions, additions or deletions.

As used herein, the terms “variant engineered nucleic acid-guided nuclease” or “variant nucleic acid-guided nuclease” refer to nucleic acid-guided nucleases have been engineered to mutate the PAM interacting domains in the LbCas12a (Lachnospiraceae bacterium Cas12a), AsCas 12a (Acidaminococcus sp. BV3L6 Cas12a), CtCas12a (Candidatus Methanoplasma termitum Cas12a), EeCas12a (Eubacterium eligens Cas12a), Mb3Cas12a (Moraxella bovoculi Cas12a), FnCas12a (Francisella novicida Cas12a), FnoCas12a (Francisella tularensis subsp. novicida FTG Cas12a), FbCas12a (Flavobacteriales bacterium Cas12a), Lb4Cas12a (Lachnospira eligens Cas12a), MbCas12a (Moraxella bovoculi Cas12a), Pb2Cas12a (Prevotella bryantii Cas12a), PgCas12a (Candidatus Parcubacteria bacterium Cas12a), AaCas12a (Acidaminococcus sp. Cas12a), BoCas12a (Bacteroidetes bacterium Cas12a), CMaCas12a (Candidatus Methanomethylophilus alvus Mx1201 Cas12a), and to-be-discovered equivalent Cas12a nucleic acid-guided nucleases such that double-stranded DNA (dsDNA) substrates bind to the variant nucleic acid-guided nuclease and are cleaved by the variant nucleic acid-guided nuclease at a slower rate than single-stranded DNA (ssDNA) substrates.

A “vector” is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and/or expressed in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, virus genomes, synthetic chromosomes, and the like.

DETAILED DESCRIPTION

The present disclosure provides compositions of matter and methods for cascade assays that detect nucleic acids. The cascade assays allow for massive multiplexing, and provide high accuracy, low cost, minimum workflow and results in less than one minute or, in some embodiments, virtually instantaneously, even at ambient temperatures of about 16-20° C. or less up to 48° C. The cascade assays described herein comprise first and second ribonucleoprotein complexes and either blocked nucleic acid molecules or blocked primer molecules. The blocked nucleic acid molecules or blocked primer molecules keep the second ribonucleoprotein complexes “locked” unless and until a target nucleic acid of interest activates the first ribonucleoprotein complex. The methods comprise the steps of providing cascade assay components, contacting the cascade assay components with a sample, and detecting a signal that is generated only when a target nucleic acid of interest is present in the sample.

Early and accurate identification of, e.g., infectious agents, microbe contamination, variant nucleic acid sequences that indicate the presence of diseases such as cancer or contamination by heterologous sources is important in order to select correct treatment; identify tainted food, pharmaceuticals, cosmetics and other commercial goods; and to monitor the environment. Nucleic acid-guided nucleases, such as Type V nucleic acid-guided nucleases, can be utilized for the detection of target nucleic acids of interest associated with diseases, food contamination and environmental threats. However, currently available nucleic acid detection such as quantitative PCR (also known as real time PCR or qPCR) or CRISPR-based detection assays such as SHERLOCK™ and DETECTR™ rely on DNA amplification, which requires time and may lead to changes to the relative proportion of nucleic acids, particularly in multiplexed nucleic acid assays. The lack of rapidity for these detection assays is due to the fact that there is a significant lag phase early in the amplification process where fluorescence above background cannot be detected. With qPCR, for example, there is a lag until the cycle threshold or Ct value, which is the number of amplification cycles required for the fluorescent signal to exceed the background level of fluorescence, is achieved and can be quantified.

The present disclosure describes a signal boost cascade assay and improvements thereto that can detect one or more target nucleic acids of interest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits in less than one minute and in some embodiments virtually instantaneously without the need for amplifying the target nucleic acid(s) of interest, thereby avoiding the drawbacks of multiplex amplification, such as primer-dimerization. As described in detail below, the cascade assays utilize signal boost mechanisms comprising various components including nucleic acid-guided nucleases, guide RNAs (gRNAs) incorporated into ribonucleoprotein complexes (RNP complexes), blocked nucleic acid molecules or blocked primer molecules, reporter moieties, and, in some embodiments, polymerases and template molecules. A particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in RNP1 (i.e., gRNA1), the cascade assay components are essentially identical no matter what target nucleic acid(s) of interest are being detected, and gRNA1 is easily programmable.

The improvements to the signal amplification or signal boost cascade assay described herein result from preventing undesired unwinding of the blocked nucleic acid molecules in the reaction mix by the second ribonucleoprotein complex (RNP2) before the blocked nucleic acid molecules are unblocked via trans-cleavage, leading to increased efficiency, reduced background, and increased signal-to-noise ratio in the cascade assay. Minimizing undesired unwinding serves two purposes. First, preventing undesired unwinding that happens not as a result of unblocking due to trans-cleavage subsequent to cis-cleavage of the target nucleic acid of interest or trans-cleavage of unblocked nucleic acid molecules—but due to other factors—leads to a “leaky” cascade assay system, which in turn leads to non-specific signal generation.

Second, preventing undesired unwinding limits non-specific interactions between the nucleic acid-guided nucleases (here, in the RNP2s) and blocked nucleic acid molecules such that only blocked nucleic acid molecules that become unblocked due to trans-cleavage activity react with the nucleic acid-guided nucleases. This “fidelity” in the cascade assay leads primarily to desired interactions and limits “wasteful” interactions where the nucleic acid-guided nucleases are essentially acting on blocked nucleic acid molecules rather than unblocked nucleic acid molecules. That is, the nucleic acid-guided nucleases are focused on desired interactions which then leads to immediate signal amplification or boost in the cascade assay.

The present disclosure provides three modalities to minimize leakiness leading to minimal false positives or higher background signal. The present disclosure demonstrates that undesired unwinding of the blocked nucleic acid molecules can be lessened substantially by 1) increasing the molar ratio of the concentration of blocked nucleic acid molecules (equivalent to a target nucleic acid molecule for the RNP2) to be equal to or greater than the molar concentration of RNP2 (e.g., the nucleic acid-guided nuclease in RNP2); 2) engineering the nucleic acid-guided nuclease used in RNP2 so as to increase the time it takes the nucleic acid-guided nuclease to recognize double-strand DNA at least two-fold and preferably three-fold or more; and/or 3) engineering the blocked nucleic acid molecules to include bulky modifications (that is, molecules with a size of about 1 nm or less).

The first modality for minimizing undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules) is to adjust the relative concentrations of the blocked nucleic acid molecules (or blocked primer molecules) and RNP2s such that the molar concentration of the blocked nucleic acid molecules (or blocked primer molecules) is equal to or greater than the molar concentration of RNP2s. Before the present disclosure, the common wisdom in performing CRISPR detection assays was to use a vast excess of nucleic acid-guided nuclease (e.g., RNP complex) to target.

In most detection assays, the quantity of the target nucleic acid of interest is not known (e.g., the detection assay is performed on a sample with an unknown concentration of target); however, in experiments conducted to determine the level of detection of two CRISPR detection assays known in the art, DETECTR™ and SHERLOCK™, the nucleic acid nuclease was present at ng/μL concentrations and the target of interest was present at very low copy numbers or at femtomolar to attamolar concentration. Thus, the present methods and reagent mixtures not only adjust the relative concentrations of the blocked nucleic acid molecules (or blocked primer molecules) and RNP2s such that the molar concentration of the blocked nucleic acid molecules (or blocked primer molecules) is equal to or greater than the molar concentration of RNP2s, but the molar concentration of RNP2s may still exceed the molar concentration of the blocked nucleic acid molecules by a lesser amount, such as where the molar concentration of RNP2s exceeds the molar concentration of blocked nucleic acid molecules (or blocked target molecules) by 100,000×, 50,000×, 25,000×, 10,000×, 5,000×, 1,000×, 500×, 100×, 50×, or 10× or less.

For example, Sun, et al. ran side-by-side comparisons of the DETECTR™ and SHERLOCK™ detection assays, using a concentration of 100 ng/μL LbCas12a in the DETECTR™ assay and a concentration of 20 ng/μL LwCas13a in the SHERLOCK™ assay, where the concentration of the target nucleic acid molecules ranged from 0 copies/μL, 0.1 copies/μL, 0.2 copies/μL, 1.0 copy/μL, 2.0 copies/μL, 5.0 copies/μL, 10.0 copies/μL, and so on up to 200.0 copies/μL. (Sun, et al., J. of Translational Medicine, 12:74 (2021).) In addition, Broughton, et al., ran the DETECTR™ assay using a concentration range of 2.5 copies/μL to 1250 copies/μL target nucleic acid molecules to 40 nM LbCas12 (see, Broughton, et al., Nat. Biotech., 38:870-74 (2020)); and Lee, et al., ran the SHERLOCK™ assay using a concentration range of 10 fM to 50 aM target nucleic acid molecules to 150 nM Cas12 (see Lee, et al., PNAS, 117(41):25722-31 (2020). Thus, the ratio of nucleic acid-guided nuclease to blocked nucleic acid molecule (e.g., target for RNP2) described herein is very different from ratios practiced in the art and this ratio has been determined to limit undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules).

In a second modality, variant nucleic acid-guided nucleases have been engineered to mutate the domains in the variants that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules in, e.g., Type V nucleic acid-guided nucleases such as the LbCas12a (Lachnospriaceae bacterium Cas12a), AsCas 12a (Acidaminococcus sp. BV3L6 Cas12a), CtCas12a (Candidatus Methanoplasma termitum Cas12a), EeCas12a (Eubacterium eligens Cas12a), Mb3Cas12a (Moraxella bovoculi Cas12a), FnCas12a (Francisella novicida Cas12a), FnoCas12a (Francisella tularensis subsp. novicida FTG Cas12a), FbCas12a (Flavobacteriales bacterium Cas12a), Lb4Cas12a (Lachnospira eligens Cas12a), MbCas12a (Moraxella bovoculi Cas12a), Pb2Cas12a (Prevotella bryantii Cas12a), PgCas12a (Candidatus Parcubacteria bacterium Cas12a), AaCas12a (Acidaminococcus sp. Cas12a), BoCas12a (Bacteroidetes bacterium Cas12a), CMaCas12a (Candidatus Methanomethylophilus alvus Mx1201 Cas12a), and other related nucleic acid-guided nucleases (e.g., homologs and orthologs of these nucleic acid-guided nucleases) also limit unwinding. These variant nucleic acid-guided nucleases have been engineered such that double-stranded DNA (dsDNA) substrates bind to and activate to the variant nucleic acid-guided nucleases slowly, but single-stranded DNA (ssDNA) substrates continue to bind and activate the variant nucleic acid-guided nuclease at a high rate. Thus, the variant nucleic acid-guided nucleases effect a “lock” on the RNP complex (here, the RNP2) vis-à-vis double-strand DNA. Locking RNP2 in this way lessens the likelihood of undesired unwinding of the blocked nucleic acid molecules as described in detail herein (see FIG. 1C and the accompanying discussion). Modifying the nucleic acid-guided nucleases to not recognize dsDNA or to recognize dsDNA is contrary to what is desired in other CRISPR-based diagnostic/detection assays.

Finally, another modality for minimizing undesired unwinding of the blocked nucleic acid molecules is to use “bulky modifications” at the 5′ and/or 3′ ends of the blocked nucleic acid molecules and/or at internal nucleic acid bases of the blocked nucleic acid molecules. Doing so creates steric hindrance at the domains of the nucleic acid-guided nuclease in RNP2 that interact with the PAM region or that interact with surrounding sequences on the blocked nucleic acid molecules, disrupting, e.g., PAM recognition in the target strand and preventing displacement of the non-target strand. Using bulky modifications is yet another path to locking RNP2 to double-strand DNA molecules thereby lessening the likelihood of undesired unwinding of the blocked nucleic acid molecules as described in detail herein (again, see FIG. 1C and the accompanying discussion). “Bulky modifications” include molecules with a size of about 1 nm or less.

FIG. 1A provides a simplified diagram demonstrating a prior art method for quantifying target nucleic acids of interest in a sample; namely, the quantitative polymerase chain reaction or qPCR, which to date may be considered the gold standard for quantitative detection assays. The difference between PCR and qPCR is that PCR is a qualitative technique that indicates the presence or absence of a target nucleic acid of interest in a sample, where qPCR allows for quantification of target nucleic acids of interest in a sample. qPCR involves selective amplification and quantitative detection of specific regions of DNA or cDNA (i.e., the target nucleic acid of interest) using oligonucleotide primers that flank the specific region(s) in the target nucleic acid(s) of interest. The primers are used to amplify the specific regions using a polymerase. Like PCR, repeated cycling of the amplification process leads to an exponential increase in the number of copies of the region(s) of interest; however, unlike traditional PCR, the increase is tracked using an intercalating dye or, as shown in FIG. 1A, a sequence-specific probe (e.g., a “Taq-man probe”) the fluorescence of which is detected in real time. RT-qPCR differs from qPCR in that a reverse transcriptase is used to first copy RNA molecules to produce cDNA before the qPCR process commences.

FIG. 1A is an overview of a qPCR assay where target nucleic acids of interest from a sample are amplified before detection. FIG. 1A shows the qPCR method 10, comprising a double-stranded DNA template 12 and a sequence specific Taq-man probe 14 comprising a region complementary to the target nucleic acid of interest 20, a quencher 16, a quenched fluorophore 18 where 22 denotes quenching between the quencher 16 and quenched fluorophore 18. Upon denaturation, the two strands of the double-stranded DNA template 12 separate into complementary single strands 26 and 28. In the next step, primers 24 and 24′ anneal to complementary single strands 26 and 28, as does the sequence-specific Taq-man probe 14 via the region complementary 20 to the complementary strand 26 of the target nucleic acid of interest. Initially the Taq-man probe is annealed to complementary strand 26 of the target region of interest intact; however, primers 24 and 24′ are extended by polymerase 30 but the Taq-man probe is not, due to the absence of a 3′ hydroxy group. Instead, the exonuclease activity of the polymerase “chews up” the Taq-man probe, thereby separating the quencher 16 from the quenched fluorophore 18 resulting in an unquenched or excited-state fluorophore 34. The fluorescence quenching ensures that fluorescence occurs only when target nucleic acids of interest are present and being copied, where the fluorescent signal is proportional to the number of single-strand target nucleic acids being amplified.

As noted above, the downside to the prior art, currently available detection assays such as qPCR, as well as CRISPR-based reaction assays such as SHERLOCK™ and DETECTR™ is that these assays rely on DNA amplification, which, in addition to issues with multiplexing, significantly hinders the ability to perform rapid testing, e.g., in the field. That is, where the present cascade assay works at ambient temperatures, including room temperatures and below, assays that require amplification of the target nucleic acids of interest do not work well at lower temperatures—even those assays utilizing isothermal amplification—due to non-specific binding of the primers and low polymerase activity. Further, primer design is far more challenging. As for the lack of rapidity of detection assays that require amplification of the target nucleic acids of interest, a significant lag phase occurs early in the amplification process where fluorescence above background cannot be detected, particularly in samples with very low copy numbers of the target nucleic acid of interest. And, again, amplification, particularly multiplex amplification, may cause changes to the relative proportion of nucleic acids in samples that, in turn, lead to artifacts or inaccurate results.

FIG. 1B provides a simplified diagram demonstrating a method (100) of a cascade assay. The cascade assay is initiated when the target nucleic acid of interest (104) binds to and activates a first pre-assembled ribonucleoprotein complex (RNP1) (102). A ribonucleoprotein complex comprises a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to the target nucleic acid of interest, guides an RNP complex to the target nucleic acid of interest and hybridizes to it. Typically, preassembled RNP complexes are employed in the reaction mix—as opposed to separate nucleic acid-guided nucleases and gRNAs—to facilitate rapid (and in the present cascade assays, virtually instantaneous) detection of the target nucleic acid(s) of interest.

“Activation” of RNP1 refers to activating trans-cleavage activity of the nucleic acid-guided nuclease in RNP1 (106) by binding of the target nucleic acid-guided nuclease to the gRNA of RNP1, initiating cis-cleavage where the target nucleic acid of interest is cleaved by the nucleic acid-guided nuclease. This binding and/or cis-cleavage activity then initiates trans-cleavage activity (i.e., multi-turnover activity) of the nucleic acid-guided nuclease, where trans-cleavage is indiscriminate, leading to non-sequence-specific cutting of nucleic acid molecules by the nucleic acid-guided nuclease of RNP1 (102). This trans-cleavage activity triggers activation of blocked ribonucleoprotein complexes (RNP2s) (108) in various ways, which are described in detail below. Each newly activated RNP2 (110) activates more RNP2 (108→110), which in turn cleave reporter moieties (112). The reporter moieties (112) may be a synthetic molecule linked or conjugated to a quencher (114) and a fluorophore (116) such as, for example, a probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The quencher (114) and fluorophore (116) can be about 20-30 bases apart (or about 10-11 nm apart) or less for effective quenching via fluorescence resonance energy transfer (FRET). Reporter moieties also are described in greater detail below.

As more RNP2s are activated (108→110), more trans-cleavage activity is activated and more reporter moieties are activated (where here, “activated” means unquenched); thus, the binding of the target nucleic acid of interest (104) to RNP1 (102) initiates what becomes a cascade of signal production (120), which increases exponentially; hence, the terms “signal amplification” or “signal boost.” The cascade assay thus comprises a single turnover event that triggers a multi-turnover event that then triggers another multi-turnover event in a “cascade.” As described below in relation to FIG. 4 , the reporter moieties (112) may be provided as molecules that are separate from the other components of the nucleic acid-guided nuclease cascade assay, or the reporter moieties may be covalently or non-covalently linked to the blocked nucleic acid molecules or synthesized activating molecules (i.e., the target molecules for the RNP2).

As described in detail below, the present description presents three modalities for minimizing undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules), which possess regions of double-strand DNA, where such unwinding can lead to non-specific signal generation and false positives. The modalities are 1) altering the ratio of the nucleic acid-guided nuclease in RNP2 to the blocked nucleic acid molecules in contravention to the common wisdom for CRISPR detection/diagnostic assays; 2) engineering the nucleic acid-guided nuclease used in RNP2 so that recognition of double-stranded DNA occurs more slowly than for single-strand DNA, in contravention to nucleic acid-guided nucleases that are used in other CRISPR-based detection assays; and 3) modifying the 5′ and/or 3′ ends and/or various internal nucleic acid bases of the blocked nucleic acid molecules. One, two or all three of these modalities may be employed in a given assay.

FIG. 1C is an illustration of the effects of unwinding. FIG. 1C shows at left a double-strand blocked nucleic acid molecule comprising a target strand and a non-target strand, where the non-target strand comprises regions (shown as loops) unhybridized to the target strand. Proceeding right at top, cleavage of the loops in the non-target strand by trans-cleavage initiated by RNP1 or RNP2 destabilizes the double-strand blocked nucleic acid molecule; that is, the now short regions of the non-target strand that are hybridized to the target strand become destabilized and dehybridize. As these short regions dehybridize, the target strand is released and can bind to gRNA2 in RNP2, triggering cis-cleavage of the target strand followed by trans-cleavage of additional blocked nucleic acid molecules. This process is the signal boost assay working as designed.

The pathway at the bottom of FIG. 1C illustrates the effect of undesired unwinding; that is, unwinding due not to trans-cleavage as designed but by other unwinding due to recognition of the blocked nucleic acid molecule by gRNA2 and the nucleic acid-guided nuclease in RNP2. As seen in the alternative pathway at bottom of FIG. 1C, R-loop formation between RNP2 and the blocked nucleic acid molecule (or blocked primer molecule) can still occur due to unwinding of the blocked nucleic acid molecule after gRNA2 identifies the PAM. Indeed, this unwinding can occur even in the absence of a PAM. It is an inherent characteristic of the biology of nucleic acid-guided nucleases.

Various components of the cascade assay, descriptions of how the cascade assays work, and the modalities used to minimize undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules) are described in detail below.

Target Nucleic Acids of Interest

The target nucleic acid of interest may be a DNA, RNA, or cDNA molecule. Target nucleic acids of interest may be isolated from a sample or organism by standard laboratory techniques or may be synthesized by standard laboratory techniques (e.g., RT-PCR). The target nucleic acids of interest are identified in a sample, such as a biological sample from a subject (including non-human animals or plants), items of manufacture, or an environmental sample (e.g., water or soil). Non-limiting examples of biological samples include blood, serum, plasma, saliva, mucus, a nasal swab, a buccal swab, a cell, a cell culture, and tissue. The source of the sample could be any mammal, such as, but not limited to, a human, primate, monkey, cat, dog, mouse, pig, cow, horse, sheep, and bat. Samples may also be obtained from any other source, such as air, water, soil, surfaces, food, beverages, nutraceuticals, clinical sites or products, industrial sites (including food processing sites) and products, plants and grains, cosmetics, personal care products, pharmaceuticals, medical devices, agricultural equipment and sites, and commercial samples.

In some embodiments, the target nucleic acid of interest is from an infectious agent (e.g., a bacteria, protozoan, insect, worm, virus, or fungus) that affects mammals, including humans. As a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from bacteria, such as Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Acinetobacter calcoaceticus-baumannii complex, Bacteroides fragilis, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group, Moraxella catarrhalis, Proteus spp., Salmonella enterica, Serratia marcescens, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Chlamydia tracomatis, Neisseria gonorrhoeae, Syphilis (Treponema pallidum), Ureaplasma urealyticum, Mycoplasma genitalium, and/or Gardnerella vaginalis. Also, as a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a virus, such as adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human metapneumovirus, human rhinovirus, enterovirus, influenza A, influenza A/H1, influenza A/_3, influenza A/11-2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus, herpes simplex virus 1, herpes simplex virus 2, human immunodeficiency virus (HIV), human papillomavirus, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and/or human parvovirus B 19 (B 19V). Also, as a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a fungus, such as Candida albicans, Candida auris, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and/or Cryptococcus gattii. As another non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a protozoan, such as Trichomonas vaginalis. See, e.g., Table 1 for an exemplary list of human pathogens, Table 2 for an exemplary list of human sexually transmissible diseases.

TABLE 1 Human Pathogens NCBI Taxonomy NCBI Sequence Name Category ID ID Number Acinetobacter baumannii Bacteria 470 GCF_008632635.1 Acinetobacter calcoaceticus Bacteria 471 GCF_002055515.1 Acinetobacter Bacteria 909768 Not applicable calcoaceticus-baumannii complex Anaplasma Bacteria 948 GCF_000439775.1 phagocytophilum Bacillus anthracis Bacteria 1392 GCF_000008445.1 Bacteroides fragilis Bacteria 817 GCF_016889925.1 Bartonella henselae Bacteria 38323 GCF_000612965.1 Bordetella parapertussis Bacteria 519 GCF_004008295.1 Bordetella pertussis Bacteria 520 GCF_004008975.1 Borrelia mayonii Bacteria 1674146 GCF_001936295.1 Borrelia miyamotoi Bacteria 47466 GCF_003431845.1 Brucella abortus Bacteria 235 GCF_000054005.1 Brucella melitensis Bacteria 29459 GCF_000007125.1 Brucella suis Bacteria 29461 GCF_000007505.1 Burkholderia mallei Bacteria 13373 GCF_002346025.1 Burkholderia pseudomallei Bacteria 28450 GCF_000756125.1 Campylobacter jejuni Bacteria 197 GCF_000009085.1 Chlamydia pneumoniae Bacteria 83558 GCF_000007205.1 Chlamydia psittaci Bacteria 83554 GCF_000204255.1 Chlamydia Tracomatis Bacteria 813 GCF_000008725.1 Clostridium botulinum Bacteria 1491 GCF_000063585.1 Clostridium perfringens Bacteria 1502 GCF_020138775.1 Coxiella burnetii Bacteria 777 GCF_000007765.2 Ehrlichia chaffeesis Bacteria 945 GCF_000632965.1 Ehrlichia ewingii Bacteria 947 Not available Ehrlichia ruminantium Bacteria 779 GCF_013460375.1 Enterobacter cloacae Bacteria 550 GCF_000770155.1 Enterobacter cloacae Bacteria 354276 Not applicable complex Enterococcus faecalis Bacteria 1351 GCF_000393015.1 Enterococcus faecium Bacteria 1352 GCF_009734005.1 Escherichia coli Bacteria 562 GCF_000008865.2 Francisella tularensis Bacteria 263 GCF_000156415.1 Gardnerella vaginalis Bacteria 2702 GCF_002861965.1 Haemophilus influenzae Bacteria 727 GCF_000931575.1 Klebsiella aerogenes Bacteria 548 GCF_007632255.1 Klebsiella oxytoca Bacteria 571 GCF_003812925.1 Klebsiella pneumoniae Bacteria 573 GCF_000240185.1 Legionella pneumophila Bacteria 446 GCF_001753085.1 Leptospira interrogans Bacteria 173 GCF_002073495.2 Leptospira kirschneri Bacteria 29507 GCF_000243695.2 Leptospira wolffii Bacteria 409998 GCF_004770635.1 Listeria monocytogenes Bacteria 1639 GCF_000196035.1 Moraxella catarrhalis Bacteria 480 GCF_002080125.1 Mycobacterium tuberculosis Bacteria 1773 GCF_000195955.2 Mycoplasma genitalium Bacteria 2097 GCF_000027325.1 Mycoplasma pneumoniae Bacteria 2104 GCF_900660465.1 Neisseria gonorrhoeae Bacteria 485 GCF_013030075.1 Neisseria meningitidis Bacteria 487 GCF_008330805.1 Proteus hauseri Bacteria 183417 GCF_004116975.1 Proteus mirabilis Bacteria 584 GCF_000069965.1 Proteus penneri Bacteria 102862 GCF_022369495.1 Proteus vulgaris Bacteria 585 GCF_000754995.1 Pseudomonas aeruginosa Bacteria 287 GCF_000006765.1 Rickettsia parkeri Bacteria 35792 GCF_005549115.1 GCA_018610945.1 GCF_000965075.1 GCF_000965085.1 GCF_000284195.1 GCF_000965145.1 Rickettsia prowazekii Bacteria 782 GCF_000277165.1 Rickettsia rickettsii Bacteria 783 GCF_000017445.4 Salmonella bongori Bacteria 54736 GCF_000439255.1 Salmonella enterica Bacteria 28901 GCF_000006945.2 Salmonella enterica Bacteria 28901 GCF_000006945.2 Serratia marcescens Bacteria 615 GCF_003516165.1 Shigella boydii Bacteria 621 GCF_001905915.1 Shigella dysenteriae Bacteria 622 GCF_001932995.2 Shigella flexneri Bacteria 623 GCF_000006925.2 Shigella sonnei Bacteria 624 GCF_013374815.1 Staphylococcus auerus Bacteria 1280 GCF_000013425.1 Staphylococcus enterotoxin Bacteria 1280 U93688.2 B Staphylococcus epidermidis Bacteria 1282 GCF_006094375.1 Staphylococcus lugdunensis Bacteria 28035 GCF_001558775.1 Stenotrophomonas Bacteria 40324 GCF_900475405.1 maltophilia Streptococcus agalactiae Bacteria 1311 GCF_001552035.1 Streptococcus pneumoniae Bacteria 1313 GCF_002076835.1 Streptococcus pyogenes Bacteria 1314 GCF_900475035.1 Treponema pallidum Bacteria 160 GCF_000246755.1 Ureaplasma urealyticum Bacteria 2130 GCF_000021265.1 Vibrio parahaemolyticus Bacteria 670 GCF_000196095.1 Vibrio vulnificus Bacteria 672 GCF_002204915.1 Yersinia enterocolitica Bacteria 630 GCF_001160345.1 Yersinia pestis Bacteria 632 GCF_000222975.1 Candida albicans Fungus 5476 GCF_000182965.3 Candida auris Fungus 498019 GCF_002775015.1 Candida glabrata Fungus 5478 GCF_000002545.3 Candida parapsilosis Fungus 5480 GCF_000182765.1 Candida tropicalis Fungus 5482 GCF_000006335.3 Coccidioides immitis Fungus 5501 GCF_000149335.2 Coccidioides posadasii Fungus 199306 GCF_000151335.2 Cokeromyces recurvatus Fungus 90255 GCA_000697235.1 Cryptococcus gattii Fungus 37769 GCF_000185945.1 Cryptococcus neoformans Fungus 5207 GCF_000091045.1 Cunninghamella Fungus 90251 GCA_000697215.1 bertholletiae Encephalitozoon cuniculi Fungus 6035 GCF_000091225.1 Encephalitozoon hellem Fungus 27973 GCF_000277815.2 Encephalitozoon intestinalis Fungus 58839 GCF_000146465.1 Enterocystozoon bieneusi Fungus 31281 GCF_000209485.1 Mortierella wolfii Fungus 90253 GCA_016098105.1 Pichia kudriavzevii Fungus 4909 GCF_003054445.1 Saksenaea vasiformis Fungus 90258 GCA_000697055.1 Syncephalastrum Fungus 13706 GCA_002105135.1 racemosum Trichomonas vaginalis Fungus 5722 GCF_000002825.2 Ricinus communis Plant 3988 GCF_019578655.1 Acanthamoeba castellanii Protozoa 5755 GCF_000313135.1 Babesia divergens Protozoa 32595 GCA_001077455.2 Babesia microti Protozoa 5868 GCF_000691945.2 Balamuthia mandrillaris Protozoa 66527 GCA_001185145.1 Cryptosporidium parvum Protozoa 5807 GCF_000165345.1 Cyclospora cayatanensis Protozoa 88456 GCF_002999335.1 Entamoeba histolytica Protozoa 5759 GCF_000208925.1 Giardia lamblia Protozoa 5741 GCF_000002435.2 Naegleria fowleri Protozoa 5763 GCF_008403515.1 Toxoplasma gondii Protozoa 5811 GCF_000006565.2 Alkhumra hemorrhagic Virus 172148 JF416961.1 fever virus Argentinian Virus 2169991 GCF_000856545.1 mammarenavirus Betacoronavirus 1 Virus 694003 GCF_000862505.1 GCF_003972325.1 Black Creek Canal Virus 1980460 GCF_002817355.1 orthohantavirus California encephalitis Virus 1933264 GCF_003972565.1 orthobunyavirus Chapare mammarenavirus Virus 499556 GCF_000879235.1 Chikungunya virus Virus 37124 GCF_000854045.1 Crimean-Congo Virus 1980519 GCF_000854165.1 hemorrhagic fever orthnairovirus Dabie bandavirus Virus 2748958 GCF_000897355.1 GCF_003087855.1 Deer tick virus Virus 58535 MZ148230 to MZ148271 Dengue virus 1 Virus 11053 GCF_000862125.1 Dengue virus 2 Virus 11060 GCF_000871845.1 Dengue virus 3 Virus 11069 GCF_000866625.1 Dengue virus 4 Virus 11070 GCF_000865065.1 Eastern equine encephalitis Virus 11021 GCF_000862705.1 virus Enterovirus A Virus 138948 GCF_002816655.1 GCF_000861905.1 GCF_001684625.1 Enterovirus B Virus 138949 GCF_002816685.1 GCF_000861325.1 Enterovirus C Virus 138950 GCF_000861165.1 Enterovirus D Virus 138951 GCF_000861205.1 GCF_002816725.1 Guanarito mammarenavirus Virus 45219 GCF_000853765.1 Heartland bandavirus Virus 2747342 GCF_000922255.1 Hendra henipavirus Virus 63330 GCF_000852685.1 Hepacivirus C Virus 11103 GCF_002820805.1 GCF_000861845.1 GCF_000871165.1 GCF_000874285.1 GCF_001712785.1 hepatitis A virus Virus 208726 K02990.1 M14707.1 M20273.1 X75215.1 AB020564.1 hepatitis B virus Virus 10407 GCF_000861825.2 hepatitis C virus Virus 11103 GCF_002820805.1 GCF_000861845.1 GCF_000871165.1 GCF_000874285.1 GCF_000874265.1 GCF_001712785.1 Hepatovirus A Virus 12092 GCF_000860505.1 Human adenovirus A Virus 129875 GCF_000846805.1 Human adenovirus B Virus 108098 GCF_000857885.1 Human adenovirus C Virus 129951 GCF_000858645.1 Human adenovirus D Virus 130310 GCF_000885675.1 Human adenovirus E Virus 130308 GCF_000897015.1 Human adenovirus F Virus 130309 GCF_000846685.1 Human adenovirus G Virus 536079 GCF_000847325.1 Human alphaherpesvirus 1 Virus 10298 GCF_000859985.2 Human alphaherpesvirus 2 Virus 10310 GCF_000858385.2 human betaherpesvirus 6A Virus 32603 GCF_000845685.2 human betaherpesvirus 6B Virus 32604 GCF_000846365.1 Human coronavirus 229E Virus 11137 GCF_001500975.1 GCF_000853505.1 Human coronavirus HKU1 Virus 290028 GCF_000858765.1 Human coronavirus NL63 Virus 277944 GCF_000853865.1 Human coronavirus OC43 Virus 31631 GCF_003972325.1 Human gammaherpesvirus Virus 37296 GCF_000838265.1 8 Human immunodeficiency Virus 11676 GCF_000864765.1 virus 1 Human immunodeficiency Virus 11709 GCF_000856385.1 virus 2 human metapneumovirus Virus 162145 GCF_002815375.1 human papillomavirus Virus GCF_001274345.1 Human polyomavirus 1 Virus 1891762 GCF_000837865.1 Human polyomavirus 2 Virus 1891763 GCF_000863805.1 human rhinovirus A Virus 147711 GCF_000862245.1 GCF_002816835.1 human rhinovirus B Virus 147712 GCF_000861265.1 GCF_002816855.1 human rhinovirus C Virus 463676 GCF_002816885.1 GCF_000872325.1 Influenza A virus Virus 11320 GCF_001343785.1 GCF_000851145.1 GCF_000866645.1 Influenza B virus Virus 11520 GCF_000820495.2 Influenza C virus Virus 11552 GCF_000856665.10 Influenza D virus Virus 1511084 GCF_002867775.1 Japanese encephalitis virus Virus 11072 GCF_000862145.1 Kyasanur Forest disease Virus 33743 GCF_002820625.1 virus La Crosse orthobunyavirus Virus 2560547 GCF_000850965.1 Lassa virus Virus 11620 GCF_000851705.1 Lujo mammarenavirus Virus 649188 GCF_000885555.1 Lyssavirus australis Virus 90961 GCF_000850325.1 Marburg virus Virus NC_001608.3 Measles morbillivirus Virus 11234 GCF_000854845.1 Middle East respiratory Virus 1335626 GCF_002816195.1 syndrome-related GCF_000901155.1 coronavirus Monongahela hantavirus Virus 2259728 MH539865 MH539866 MH539867 New York hantavirus Virus 44755 U36803.1 U36802.1 U36801.1 U09488.1 Nipah henipavirus Virus 121791 GCF_000863625.1 Norwalk virus Virus 11983 GCF_000864005.1 GCF_008703965.1 GCF_008703985.1 GCF_008704025.1 GCF_010478905.1 GCF_000868425.1 Omsk hemorrhagic fever Virus 12542 GCF_000855505.1 virus parainfluenza virus 1 Virus 12730 GCF_000848705.1 NC_003461 parainfluenza virus 2 Virus X57559.1 AF533010 AF533011 AF533012 parainfluenza virus 3 Virus 11216 GCA_006298365.1 GCA_000850205.1 parainfluenza virus 4 Virus 2560526 NC_021928.1 Paslahepevirus balayani Virus 1678141 GCF_000861105.1 Poliovirus Virus 138950 GCF_000861165.1 Primate erythroparvovirus 1 Virus 1511900 GCF_000839645.1 Rabies lyssavirus Virus 11292 GCF_000859625.1 respiratory syncytial virus Virus 12814 GCF_000856445.1 Rift Valley virus Virus 11588 HE687302 HE687307 Saint Louis encephalitis Virus 11080 GCF_000866785.1 virus Sapporo virus Virus 95342 GCF_000849945.1 GCF_000855765.1 GCF_000854265.1 GCF_001008475.1 GCF_000853825.1 SARS-related coronavirus Virus 694009 GCF_000864885.1 GCF_009858895.2 Severe acute respiratory Virus 2901879 NC_004718.3 syndrome coronavirus 1 Severe acute respiratory Virus 2697049 NC_045512.2 syndrome coronavirus 2 Sin Nombre virus Virus 1980491 GCF_000854765.1 Tick-borne encephalitis Virus 11084 GCF_000863125.1 virus Variola major Virus 12870 not available Variola minor Virus 53258 not available Variola virus Virus 10255 GCF_000859885.1 Venezuelan equine Virus 11036 GCF_000862105.1 encephalitis virus West Nile virus Virus 11082 GCF_000861085.1 GCF_000875385.1 Western equine encephalitis Virus 11039 GCF_000850885.1 virus Yellow fever virus Virus 11089 GCF_000857725.1 Zaire ebolavirus Virus 186538 GCF_000848505.1 Zika virus Virus 64320 GCF_000882815.3 GCF_002366285.1

TABLE 2 Human STD pathogens NCBI NCBI Taxonomy Sequence ID Name Category ID Number Pthirus pubis Animal 121228 MT721740.1 Sarcoptes scabiei Animal 52283 GCA_020844145.1 Chlamydia trachomatis Bacteria 813 GCF_000008725.1 Gardnerella vaginalis Bacteria 2702 GCF_002861965.1 Haemophilus ducreyi Bacteria 730 GCF_001647695.1 Mycoplasma genitalium Bacteria 2097 GCF_000027325.1 Neisseria gonorrhoeae Bacteria 485 GCF_013030075.1 Treponema pallidum Bacteria 160 GCF_000246755.1 Trichomonas vaginalis Protozoa 5722 GCF_000002825.2 Hepacivirus C Virus 11103 GCF_002820805.1 Hepatitis B virus Virus 10407 GCF_000861825.2 Hepatitis delta virus Virus 12475 GCF_000856565.1 Hepatovirus A Virus 12092 GCF_000860505.1 Human alphaherpesvirus 1 Virus 10298 GCF_000859985.2 Human immunodeficiency Virus 11676 GCF_000864765.1 virus 1 Human immunodeficiency Virus 11709 GCF_000856385.1 virus 2 Human papillomavirus Virus 10566 GCF_001274345.1

Additionally, the target nucleic acid of interest may originate in an organism such as a bacterium, virus, fungus or other pest that infects livestock or agricultural crops. Such organisms include avian influenza viruses, mycoplasma and other bovine mastitis pathogens, Clostridium perfringens, Campylobacter sp., Salmonella sp., Pospirivoidae, Avsunvirodiae, Pantoea stewartii, Mycoplasma genitalium, Spiroplasma sp., Pseudomonas solanacearum, Erwinia amylovora, Erwinia carotovora, Pseudomonas syringae, Xanthomonas campestris, Agrobacterium tumefaciens, Spiroplasma citri, Phytophthora infestans, Endothia parasitica, Ceratocysis ulmi, Puccinia graminis, Hemilea vastatrix, Ustilage maydis, Ustilage nuda, Guignardia bidweliji, Uncinula necator, Botrytis cincerea, Plasmopara viticola, or Botryotinis fuckleina. See, e.g., Table 3 for an exemplary list of non-human animal pathogens.

TABLE 3 Animal Pathogens NCBI NCBI Sequence ID Name Category Taxonomy ID Number Acarapis woodi Animal 478375 GCA_023170135.1 Aethina tumida Animal 116153 GCF_001937115.1 Chorioptes bovis Animal 420257 Chrysomya bezziana Animal 69364 Cochliomyia hominivorax Animal 115425 GCA_004302925.1 Echinococcus granulosus Animal 6210 GCF_000524195.1 Echinococcus Animal 6211 GCA_000469725.3 multilocularis Gyrodactylus salaris Animal 37629 GCA_000715275.1 Psoroptes ovis Animal 83912 GCA_002943765.1 Sarcoptes scabiei Animal 52283 GCA_020844145.1 Taenia solium Animal 6204 GCA_001870725.1 Trichinella britovi Animal 45882 GCA_001447585.1 Trichinella nativa Animal 6335 GCA_001447565.1 Trichinella nelsoni Animal 6336 GCA_001447455.1 Trichinella papuae Animal 268474 GCA_001447755.1 Trichinella pseudospiralis Animal 6337 GCA_001447645.1 Trichinella spiralis Animal 6334 GCF_000181795.1 Trichinella zimbabwensis Animal 268475 GCA_001447665.1 Tropilaelaps clareae Animal 208209 Tropilaelaps koenigerum Animal 208208 Tropilaelaps mercedesae Animal 418985 GCA_002081605.1 Tropilaelaps thaii Animal 418986 Varroa destructor Animal 109461 GCF_002443255.1 Varroa jacobsoni Animal 62625 GCF_002532875.1 Varroa rindereri Animal 109259 Varroa underwoodi Animal 109260 Anaplasma centrale Bacteria 769 GCF_000024505.1 Anaplasma marginale Bacteria 770 GCF_000020305.1 Bacillus anthracis Bacteria 1392 GCF_000008445.1 Brucella abortus Bacteria 235 GCF_000054005.1 Brucella melitensis Bacteria 29459 GCF_000007125.1 Brucella ovis Bacteria 236 GCF_000016845.1 Brucella suis Bacteria 29461 GCF_000007505.1 Burkholderia mallei Bacteria 13373 GCF_002346025.1 Burkholderia pseudomallei Bacteria 28450 GCF_000756125.1 Campylobacter fetus Bacteria 196 GCF_000015085.1 Candidatus Xenohaliotis Bacteria 84677 californiensis Candidatus Hepatobacter Bacteria 1274402 GCF_000742475.1 penaei Chlamydia abortus Bacteria 83555 GCF_900416725.2 Chlamydia psittaci Bacteria 83554 GCF_000204255.1 Corynebacterium Bacteria 1719 GCF_001865765.1 pseudotuberculosis Coxiella burnetii Bacteria 777 GCF_000007765.2 Ehrlichia ruminantium Bacteria 779 GCF_013460375.1 Francisella tularensis Bacteria 263 GCF_000156415.1 Melissococcus plutonius Bacteria 33970 GCF_003966875.1 Mycobacterium avium Bacteria 1764 GCF_000696715.1 Mycobacterium Bacteria 1773 GCF_000195955.2 tuberculosis Mycoplasma capricolum Bacteria 2095 GCF_000012765.1 Mycoplasma gallisepticum Bacteria 2096 GCF_000286675.1 Mycoplasma mycoides Bacteria 2102 GCF_000023685.1 Mycoplasma putrefaciens Bacteria 2123 GCF_900476175.1 Mycoplasmopsis agalactiae Bacteria 2110 GCF_009150585.1 Mycoplasmopsis synoviae Bacteria 2109 GCF_013393745.1 Paenibacillus larvae Bacteria 1464 GCF_002951935.1 Pasteurella multocida Bacteria 747 GCF_000006825.1 Salmonella enterica Bacteria 28901 GCF_000006945.2 Streptococcus equi Bacteria 1336 GCF_015689455.1 Taylorella equigenitalis Bacteria 29575 GCF_002288025.1 Vibrio parahaemolyticus Bacteria 670 GCF_000196095.1 Batrachochytrium Fungi 109871 GCF_000203795.1 dendrobatidis Batrachochytrium Fungi 1357716 GCA_021556675.1 salamandrivorans Aphanomyces astaci Oomycota 112090 GCF_000520075.1 Aphanomyces invadans Oomycota 157072 GCF_000520115.1 Babesia bigemina Protozoa 5866 GCF_000981445.1 Babesia bovis Protozoa 5865 GCA_000165395.2 Babesia caballi Protozoa 5871 Bonamia exitiosa Protozoa 362532 Bonamia ostreae Protozoa 126728 Leishmania amazonensis Protozoa 5659 GCA 005317125.1 Leishmania braziliensis Protozoa 5660 GCF_000002845.2 Leishmania donovani Protozoa 5661 GCF_000227135.1 Leishmania infantum Protozoa 5671 GCF_000002875.2 Leishmania major Protozoa 5664 GCF_000002725.2 Leishmania mexicana Protozoa 5665 GCF_000234665.1 Leishmania tropica Protozoa 5666 GCA_014139745.1 Marteilia refringens Protozoa 107386 Perkinsus marinus Protozoa 31276 GCF_000006405.1 Perkinsus olseni Protozoa 32597 GCA_013115135.1 Theileria annulata Protozoa 5874 GCF_000003225.4 Theileria equi Protozoa 5872 GCF_000342415.1 Theileria parva Protozoa 5875 GCF_000165365.1 Tritrichomonas foetus Protozoa 1144522 GCA_001839685.1 Trypanosoma brucei Protozoa 5691 GCF_000002445.2 Trypanosoma congolense Protozoa 5692 GCA_002287245.1 Trypanosoma equiperdum Protozoa 5694 GCA_001457755.2 Trypanosoma evansi Protozoa 5697 GCA_917563935.1 Trypanosoma vivax Protozoa 5699 GCA_021307395.1 African horse Virus 40050 GCF_000856125.1 sickness virus African swine fever virus Virus 10497 GCF_000858485.1 Akabane orthobunyavirus Virus 1933178 GCF_000871205.1 Alcelaphine Virus 35252 GCF_000838825.1 gammaherpesvirus 1 Alphaarterivirus equid Virus 2499620 GCF_000860865.1 Alphacoronavirus 1 Virus 693997 GCF_000856025.1 Ambystoma tigrinum virus Virus 265294 GCF_000841005.1 Avian coronavirus Virus 694014 GCF_012271565.1 Avian influenza virus Virus 11309 Avian metapneumovirus Virus 38525 GCF_002989735.1 Avian orthoavulavirus 1 Virus 2560319 GCF_002834085.1 Avihepatovirus A Virus 691956 GCF_000869945.1 Betaarterivirus suid 1 Virus 2499680 GCF_003971765.1 Bluetongue virus Virus 40051 GCF_000854445.3 Bovine alphaherpesvirus 1 Virus 10320 GCF_008777455.1 Bovine leukemia virus Virus 11901 GCF_000853665.1 Camelpox virus Virus 28873 GCF_000839105.1 Caprine arthritis Virus 11660 GCF_000857525.1 encephalitis virus Crimean-Congo Virus 1980519 GCF_000854165.1 hemorrhagic fever orthonairovirus Cyprinid herpesvirus 3 Virus 180230 GCF_000871465.1 Decapod iridescent virus 1 Virus 2560405 GCF_004788555.1 Decapod Virus 1513224 GCF_000844705.1 penstyldensovirus 1 Deformed wing virus Virus 198112 GCF_000852585.1 Eastern equine Virus 11021 GCF_000862705.1 encephalitis virus Epizootic haematopoietic Virus 100217 GCF_001448375.1 necrosis virus Epizootic hemorrhagic Virus 40054 GCF_000885335.1 disease virus Equid alphaherpesvirus 1 Virus 10326 GCF_000844025.1 Equid alphaherpesvirus 4 Virus 10331 GCF_000846345.1 Equine infectious Virus 11665 GCF_000847605.1 anemia virus Foot-and-mouth disease Virus 12110 GCF_002816555.1 virus Frog virus 3 Virus 10493 GCF_002826565.1 Gallid alphaherpesvirus 1 Virus 10386 GCF_000847005.1 Goatpox virus Virus 186805 GCF_000840165.1 Haliotid herpesvirus 1 Virus 1513231 GCF_000900375.1 Hendra henipavirus Virus 63330 GCF_000852685.1 Infectious bursal Virus 10995 GCF_000855485.1 disease virus Infectious spleen Virus 180170 GCF_000848865.1 and kidney necrosis virus Influenza A virus Virus 11320 GCF_000851145.1 Isavirus salaris Virus 55987 GCF_000854145.2 Japanese encephalitis virus Virus 11072 GCF_000862145.1 Lumpy skin disease virus Virus 59509 GCF_000839805.1 Lyssavirus rabies Virus 11292 GCF_000859625.1 Macrobrachium Virus 222557 GCA_000856985.1 rosenbergii nodavirus Middle East respiratory Virus 1335626 GCF_002816195.1 syndrome-related coronavirus Myxoma virus Virus 10273 GCF_000843685.1 Nairobi sheep Virus 1980526 GCF_002117695.1 disease orthonairovirus Nipah henipavirus Virus 121791 GCF_000863625.1 Norwegian salmonid Virus 344701 alphavirus Novirhabdovirus piscine Virus 1980916 GCF_000856505.1 Novirhabdovirus salmonid Virus 1980917 GCF_000850065.1 Penaeid shrimp infectious Virus 282786 GCA_000866305.1 myonecrosis virus Peste des petits ruminants Virus 2593991 GCF_000866445.1 virus Pestivirus C Virus 2170082 GCF_000864685.1 GCF_003034095.1 Pestivirus A Virus 2170080 GCF_000861245.1 Rabbit hemorrhagic Virus 11976 GCF_000861285.1 disease virus Rift Valley fever Virus 1933187 GCF_000847345.1 phlebovirus Rinderpest morbillivirus Virus 11241 GCF_000856645.1 Severe acute Virus 694009 GCF_000864885.1 respiratory syndrome- related coronavirus Sheeppox virus Virus 10266 GCF_000840205.1 Slow bee paralysis virus Virus 458132 GCF_000887395.1 Sprivirus cyprinus Virus 696863 GCF_000850305.1 Suid alphaherpesvirus 1 Virus 10345 GCF_000843825.1 Swine vesicular Virus 12075 disease virus Taura syndrome virus Virus 142102 GCF_000849385.1 Tilapinevirus tilapiae Virus 2034996 GCF_001630085.1 Venezuelan equine Virus 11036 GCF_000862105.1 encephalitis virus Vesiculovirus indiana Virus 1972577 GCF_000850045.1 Visna-maedi virus Virus 2169971 GCF_000849025.1 West Nile Virus Virus 11082 GCF_000861085.1 Western equine Virus 11039 GCF_000850885.1 encephalitis virus White spot syndrome virus Virus 342409 GCF_000848085.2 Yellow head virus Virus 96029 GCF_003972805.1

In some embodiments, other target nucleic acids of interest may be for non-infectious conditions, e.g., to be used for genotyping, including non-invasive prenatal diagnosis of, e.g., trisomies, other chromosomal abnormalities, and known genetic diseases such as Tay Sachs disease and sickle cell anemia. Other target nucleic acids of interest and samples are described herein, such as human biomarkers for cancer. An exemplary list of human biomarkers is in Table 4. Target nucleic acids of interest may include engineered biologics, including cells such as CAR-T cells, or target nucleic acids of interest from very small or rare samples, where only small volumes are available for testing.

TABLE 4 Human Biomarkers NCBI NCBI Gene Biomarker Disease Sample Taxonomy ID ID Aß42, amyloid beta- Alzheimer disease CSF 9606 351 protein prion protein Alzheimer disease, prion CSF 9606 5621 disease Vitamin D binding multiple sclerosis CSF 9606 2638 protein progression CXCL13 multiple sclerosis CSF 9606 10563 alpha-synuclein parkinsonian disorders CSF 9606 6622 tau protein parkinsonian disorders CSF 9606 4137 Apo II parkinsonian disorders CSF 9606 336 ceruloplasmin parkinsonian disorders CSF 9606 1356 peroxisome parkinsonian disorders CSF 9606 5467 proliferation- activated PD receptor parkin neurogenerative CSF 9606 5071 disorders PTEN induced neurogenerative CSF 9606 65018 putative kinase I disorders DJ-1 (PARK7) neurogenerative CSF 9606 11315 disorders leucine-rich repeat neurogenerative CSF 9606 120892 kinase disorders secretogranin II bipolar disorder CSF 9606 7857 neurofilament light axonal degeneration CSF 9606 4747 chain IL-12B, CXDL13, Intrathecal inflammation CSF 9606 3593, 10563, IL-8 3576 ACE2 cardiovascular disease blood 9606 59272 alpha-amylase cardiovascular disease saliva 9606 276 alpha-feto protein pregnancy blood 9606 174 albumin urine diabetes 9606 213 albumin, urea albuminuria urine 9606 213 neutrophil gelatinase- acute kidney injury urine 9606 3934 associated lipocalin (NGAL) IL-18 acute kidney injury urine 9606 3606 liver fatty acid acute kidney injury urine 9606 2168 binding protein Dkk-3 prostate cancer semen 9606 27122 autoantibody to early diagnosis blood 9606 CD25 esophageal squamous cell carcinoma hTERT lung cancer blood 9606 7015 CA125 (MUC16) lung cancer blood 9606 94025 VEGF lung cancer blood 9606 7422 IL-2 lung cancer blood 9606 3558 osteopontin lung cancer blood 9606 6696 BRAF, CCNI, EGRF, lung cancer saliva 9606 673, 16007, FGF19, FRS2, 1956, GREB1, and LZTS1 9965, 10818, 9687, 11178 human epididymis ovarian cancer blood 9606 10406 protein 4 CA125 ovarian cancer saliva 9606 94025 EMP1 nasopharyngeal saliva 9606 13730 carcinoma IL-8 oral cancer saliva 9606 3576 carcinoembryonic oral or salivary saliva 9606 1048 antigen malignant tumors thioredoxin Spinalcellular carcinoma saliva 9606 7295 AIP (aryl Acute intermittent blood 9606 9049 hydrocarbon receptor porphyria, somatotroph interacting protein) adenoma, prolactin- producing pituitary gland adenoma ALK receptor Neuroblastoma blood 9606 238 tyrosine kinase susceptibility, large cell lymphoma BAP1 (BRCA1 BAP1-related tumor blood 9606 8314 associated protein 1) predisposition, melanoma susceptibility BLM Bloom syndrome blood 9606 641 BRCA1 Breast-ovarian cancer blood 9606 672 susceptibility, familial breast cancer BRCA2 Breast-ovarian cancer blood 9606 675 susceptibility, familial breast cancer, glioma susceptibility CASR (calcium Epilepsy susceptibility blood 9606 846 sensing receptor) CDC73 Hyperparathyroidism 2 blood 9606 79577 with jaw tumors CEBPA Acute myloid leukemia blood 9606 1050 EPCAM Colorectal cancer blood 9606 4072 FH hypercholesterolemia blood 9606 2271 GATA2 Acute myeloid leukemia blood 9606 2642 MITF Melanoma susceptibility blood 9606 4286 MSH2 Lynch syndrome blood 9606 4436 MSH3 Endometrial carcinoma blood 9606 4437 MSH6 Endometrial carcinoma, blood 9606 2956 colorectal cancer NF1 Neurofibromatosis, blood 9606 4763 juvenile myelomonocytic leukemia PDGRA Eosinophilic leukemia, blood 9606 5156 recurrent inflammatory gastrointestinal fibroids PHOX2B Neuroblastoma blood 9606 8929 susceptibility POT1 Melanoma blood 9606 25913 susceptibility, glioma susceptibility

The target nucleic acids of interest may be taken from environmental samples. A list of exemplary biosafety pathogens is in Table 5, and an exemplary list of known viruses is in Table 6.

TABLE 5 Exemplary Laboratory Biosafety Parasites and Pathogens NCBI NCBI Taxonomy Taxonomy Name Category ID Name Category ID Acarapis woodi Animal 478375 Streptococcus Bacteria 1349 uberis Aethina tumida Animal 116153 Besnoitia besnoiti Chromista 94643 Alaria americana Animal 2282137 Bonamia exitiosa Chromista 362532 Amblyomma Animal 6943 Bonamia ostreae Chromista 126728 americanum Amblyomma Animal 34609 Amniculicola Fungus 2566060 maculatum longissima Amphimerus Animal Arthroderma Fungus 1592210 pseudofelineus amazonicum Ancylostoma Animal 369059 Aschersonia Fungus 370936 braziliense hypocreoidea Ancylostoma Animal 29170 Aspergillago Fungus 41064 caninum clavatoflava Ancylostoma Animal 51022 Aspergillus Fungus 1904037 duodenale acidohumus Anisakis Animal 303229 Aspergillus acidus Fungus 1069201 pegreffii Anisakis simplex Animal 6269 Aspergillus Fungus 487661 aculeatinus Baylisascaris Animal 575210 Aspergillus Fungus 5053 columnaris aculeatus Baylisascaris Animal Aspergillus aeneus Fungus 41754 melis Baylisascaris Animal 6259 Aspergillus affinis Fungus 1070780 procyonis Bunostomum Animal 577651 Aspergillus Fungus 657433 phlebotomum alabamensis Ceratonova Animal 60662 Aspergillus Fungus 209559 shasta alliaceus Chrysomya Animal 69364 Aspergillus Fungus 710228 bezziana amazonicus Cochliomyia Animal 115425 Aspergillus Fungus 176160 hominivorax ambiguus Dicrocoelium Animal 57078 Aspergillus Fungus 1220191 dendriticum amoenus Diphyllobothrium Animal 28845 Aspergillus Fungus 296546 dendriticum amyloliquefaciens Diphyllobothrium Animal 60516 Aspergillus Fungus 176161 latum amylovorus Echinococcus Animal Aspergillus Fungus 2783700 granulosa angustatus Echinococcus Animal 6211 Aspergillus Fungus 454240 multilocularis anomalus Echinococcus Animal 6212 Aspergillus Fungus 37233 oligarthrus anthodesmis Echinococcus Animal 260967 Aspergillus Fungus 478867 shiquicus apicalis Echinococcus Animal 6213 Aspergillus Fungus 1140386 vogeli appendiculatus Echinostoma Animal 1873862 Aspergillus Fungus 656916 cinetorchis arachidicola Echinostoma Animal 48216 Aspergillus Fungus 1458899 hortense ardalensis Echinostoma liei Animal 48214 Aspergillus arvii Fungus 368784 Echinostoma Animal 48217 Aspergillus Fungus 1695225 revolutum askiburgiensis Fasciola hepatica Animal 6192 Aspergillus Fungus 176163 asperescens Fascioloides Animal 394415 Aspergillus Fungus 1245746 magna assulatus Gyrodactylus Animal 37629 Aspergillus Fungus 1810904 salaris astellatus Ixodes pacificus Animal 29930 Aspergillus Fungus 41725 aurantiobrunneus Ixodes ricinus Animal 34613 Aspergillus Fungus 2663348 aurantiopurpureus Ixodes scapularis Animal 6945 Aspergillus Fungus 41755 aureolatus Metagonimus Animal 84529 Aspergillus Fungus 41288 yokogawai aureoterreus Metorchis Animal Aspergillus aureus Fungus 309747 conjunctus Myxobolus Animal 59783 Aspergillus Fungus 138274 cerebralis auricomus Nanophyetus Animal 240278 Aspergillus Fungus 1250384 salmincola australiensis Necator Animal 51031 Aspergillus Fungus 1220192 americanus austroafricanus Oestrus ovis Animal 123737 Aspergillus Fungus 36643 avenaceus Opisthorchis Animal 147828 Aspergillus Fungus 105351 felineus awamori Opisthorchis Animal 6198 Aspergillus Fungus 2070749 viverrini baarnensis Parafilaria Animal 2282233 Aspergillus Fungus 1194636 bovicola baeticus Paragonimus Animal 100269 Aspergillus Fungus 522521 kellicotti bahamensis Paragonimus Animal 59628 Aspergillus Fungus 1226010 miyazakii, bertholletiae Paragonimus Animal 34504 Aspergillus Fungus 176164 westermani biplanus Psoroptes ovis Animal 83912 Aspergillus Fungus 41753 bisporus Rhipicephalus Animal 34611 Aspergillus Fungus 109264 annulatus bombycis Rhipicephalus Animal 34632 Aspergillus Fungus 1810893 sanguineus botswanensis Sarcoptes scabiei Animal 52283 Candida albicans Fungus 5476 Taenia multiceps Animal 94034 Candida glabrata Fungus 5478 Taenia saginata Animal 6206 Candida krusei Fungus 4909 Taenia solium Animal 6204 Candida Fungus 5480 parapsilosis Toxocara canis Animal 6265 Candida tropicalis Fungus 5482 Toxocara cati Animal 6266 Cryptococcus Fungus 37769 gattii Trichinella Animal 6334 Cryptococcus Fungus 5207 spiralis neoformans Trichuris suis Animal 68888 Epidermophyton Fungus 34391 floccosum Trichuris Animal 36087 Epidermophyton Fungus 74042 trichiura stockdaleae Trichuris vulpis Animal 219738 Fusarium acaciae Fungus Tropilaelaps Animal 208209 Fusarium acaciae- Fungus 282272 clareae mearnsii Tropilaelaps Animal 418985 Fusarium acicola Fungus mercedesae Uncinaria Animal 125367 Fusarium Fungus stenocephala acremoniopsis Varroa destructor Animal 109461 Fusarium Fungus acridiorum Actinobacillus Bacteria 715 Fusarium acutatum Fungus 78861 pleuropneumoniae Aeromonas Bacteria 644 Fusarium Fungus hydrophila aderholdii Aeromonas Bacteria 645 Fusarium Fungus salmonicida adesmiae Aliarcobacter Bacteria 28197 Fusarium Fungus butzleri aduncisporum Aliarcobacter Bacteria 28198 Fusarium aecidii- Fungus cryaerophilus tussilaginis Aliarcobacter Bacteria 28200 Fusarium Fungus skirrowii aeruginosam Anaplasma Bacteria 769 Fusarium Fungus 569394 centrale aethiopicum Anaplasma Bacteria 770 Fusarium affine Fungus marginale Anaplasma Bacteria 948 Fusarium Fungus phagocytophilum agaricorum Bacillus anthracis Bacteria 1392 Fusarium ril Fungus ailanthinum Bacillus cereus Bacteria 1396 Fusarium Fungus alabamense Bartonella Bacteria 38323 Fusarium albedinis Fungus henselae Bibersteinia Bacteria 47735 Fusarium albertii Fungus trehalosi Borrelia Bacteria 139 Fusarium Fungus burgdorferi albidoviolaceum Brucella abortus Bacteria 235 Fusarium albiziae Fungus Brucella canis Bacteria 36855 Fusarium Fungus albocarneum Brucella Bacteria 29459 Fusarium album Fungus melitensis Brucella ovis Bacteria 236 Fusarium Fungus aleurinum Brucella suis Bacteria 29461 Fusarium aleyrodis Fungus Burkholderia Bacteria 13373 Fusarium Fungus mallei alkanophilum Burkholderia Bacteria 28450 Fusarium Fungus pseudomallei allescheri Campylobacter Bacteria 195 Fusarium Fungus coli allescherianum Campylobacter Bacteria 32019 Fusarium allii- Fungus fetus fetus sativi Campylobacter Bacteria 32020 Trichophyton simii Fungus 63406 fetus venerealis Campylobacter Bacteria 197 Trichophyton Fungus 69891 jejuni soudanense Chlamydia Bacteria 83557 Trichophyton Fungus 34387 caviae tonsurans Chlamydia felis Bacteria 83556 Trichophyton Fungus 63417 verrucosum Chlamydia Bacteria 83560 Trichophyton Fungus 34388 muridarum violaceum Chlamydia Bacteria 85991 Ochroma Plant 66662 pecorum pyramidale Chlamydia Bacteria 83558 Babesia bigemina Protozoa 5866 pneumoniae Chlamydia Bacteria 83554 Babesia bovis Protozoa 5865 psittaci Chlamydia suis Bacteria 83559 Babesia divergens Protozoa 32595 Chlamydia Bacteria 813 Babesia jakimovi Protozoa trachomatis Chlamydophilus Bacteria Babesia major Protozoa 127461 abortus Clostridium Bacteria 1491 Babesia occultans Protozoa 536930 botulinum Clostridium Bacteria 1496 Babesia ovata Protozoa 189622 difficile Clostridium Bacteria Cryptosporidium Protozoa 5807 perfringens parvum Types A, B, C, and D Coxiella burnetii Bacteria 777 Eimeria acervulina Protozoa 5801 Cronobacter Bacteria 28141 Eimeria brunetti Protozoa 51314 sakazakii Ehrlichia canis Bacteria 944 Eimeria maxima Protozoa 5804 Ehrlichia Bacteria 945 Eimeria Protozoa 1431345 chaffeensis meleagridis Ehrlichia ewingii Bacteria 947 Eimeria necatrix Protozoa 51315 Ehrlichia ondiri Bacteria Eimeria tenella Protozoa 5802 Ehrlichia Bacteria 779 Entamoeba Protozoa 5759 ruminantium histolytica Escherichia coli Bacteria 562 Giardia duodenalis Protozoa 5741 Klebsiella Bacteria 548 Giardia lambia Protozoa aerogenes Klebsiella Bacteria 39824 Histomonas Protozoa 135588 granulomatis meleagridis Klebsiella Bacteria 2058152 Ichthyobodo Protozoa 155203 grimontii necator Klebsiella Bacteria 2153354 Ichthyophthirius Protozoa 5932 huaxiensis multifiliis Klebsiella Bacteria 2042302 Isospora burrowsi Protozoa kielensis Klebsiella Bacteria 1134687 Isospora canis Protozoa 1662860 michiganensis Klebsiella Bacteria 223378 Isospora felis Protozoa 482539 milletis Klebsiella Bacteria 571 Isospora neorivolta Protozoa oxytoca Klebsiella Bacteria 573 Isospora ohioensis Protozoa 279926 pneumoniae Klebsiella Bacteria 1463165 Leishmania Protozoa 5660 quasipneumoniae braziliensis Klebsiella Bacteria 2026240 Leishmania Protozoa 44271 quasivariicola chagasi Klebsiella Bacteria 223379 Leishmania Protozoa 5671 senegalensis infantum Klebsiella Bacteria 1641362 Marteilia Protozoa 107386 steroids refringens Klebsiella Bacteria 244366 Mikrocytos Protozoa 195010 variicola mackini Proteus mirabilis Bacteria 584 Perkinsus marinus Protozoa 31276 Pseudomonas Bacteria 89065 Perkinsus olensi Protozoa abietaniphila Pseudomonas Bacteria 407029 Sarcocystis cruzi Protozoa 5817 acephalitica Pseudomonas Bacteria 1912599 Sarcocystis hirsuta Protozoa 61649 acidophila Pseudomonas Bacteria 1302376 Sarcocystis Protozoa 61650 adelgestsugas hominis Pseudomonas Bacteria 287 Theileria annulata Protozoa 5874 aeruginosa Pseudomonas Bacteria 1387231 Theileria buffei Protozoa aestus Pseudomonas Bacteria 46677 Theileria Protozoa 77054 agarici lestoquardi Pseudomonas Bacteria Theileria Protozoa 540482 akappageensis luwenshuni Pseudomonas Bacteria 43263 Theileria mutans Protozoa 27991 alcaligenes Pseudomonas Bacteria 101564 Theileria orientalis Protozoa 68886 alcaliphila Pseudomonas Bacteria 37638 Theileria parva Protozoa 5875 alginovora Pseudomonas Bacteria Theileria sergenti Protozoa 5877 alkanolytica Pseudomonas Bacteria 237609 Theileria Protozoa 507731 alkylphenolica uilenbergi Pseudomonas Bacteria 2740531 Toxoplasma Protozoa 5811 allii gondii Pseudomonas Bacteria 2810613 Trichomonas fetus Protozoa alliivorans Pseudomonas Bacteria 2774460 Trichomonas Protozoa 56777 allokribbensis gallinae Pseudomonas Bacteria 1940621 Trichomonas Protozoa 1440121 alloputida stableri Pseudomonas Bacteria 2842348 Trypanosoma Protozoa 5691 alvandae brucei Pseudomonas Bacteria 47877 Trypanosoma Protozoa 5692 amygdali congolense Pseudomonas Bacteria 32043 Trypanosoma Protozoa 5693 amyloderamosa cruzi Pseudomonas Bacteria 2710589 Abras virus Virus 2303487 anatoliensis Pseudomonas Bacteria 147728 Absettarov virus Virus andersonii Pseudomonas Bacteria 53406 Abu Hammad Virus 248058 anguilliseptica virus Pseudomonas Bacteria 219572 Abu Mina virus Virus 248059 antarctica Pseudomonas Bacteria 485870 Acado virus Virus anuradhapurensis Pseudomonas Bacteria 2710591 Acara virus Virus 2748201 arcuscaelestis Pseudomonas Bacteria 289370 Achiote virus Virus 2036702 argentinensis Pseudomonas Bacteria 702115 Adana virus Virus 1611877 arsenicoxydans Pseudomonas Bacteria 2842349 Adelaide River Virus 31612 asgharzadehiana virus Pseudomonas Bacteria 2219225 Adria virus Virus asiatica Pseudomonas Bacteria 53407 Aedes aegypti Virus 186156 asplenii densovirus Pseudomonas Bacteria 1190415 Aedes albopictus Virus 35338 asturiensis densovirus Pseudomonas Bacteria 1825787 Aedes flavivirus Virus 390845 asuensis Pseudomonas Bacteria 2565368 Aedes galloisi Virus 1046551 atacamensis flavivirus Pseudomonas Bacteria 2609964 Aedes Virus atagonensis pseudoscutellaris densovirus Pseudomonas Bacteria 86192 Aedes Virus 341721 aurantiaca pseudoscutellaris reovirus Pseudomonas Bacteria 587851 Aedes vexans Virus 7163 aureofaciens Pseudomonas Bacteria 46257 African horse Virus 40050 avellanae sickness virus Pseudomonas Bacteria 1869229 African swine Virus 10497 aylmerensis fever virus Pseudomonas Bacteria 2843612 Aguacate virus Virus 1006583 azadiae Pseudomonas Bacteria Aino virus Virus 11582 azerbaijanoccide ntalis Pseudomonas Bacteria Akabane virus Virus 70566 azerbaijanorientalis Pseudomonas Bacteria 291995 Alajuela virus Virus 1552846 azotifigens Pseudomonas Bacteria 47878 Alcelaphine Virus 35252 azotoformans gammaherpesvirus 1 Pseudomonas Bacteria 674054 Alenquer virus Virus 629726 baetica Pseudomonas Bacteria 74829 Aleutian Mink Virus balearica Disease Pseudomonas Bacteria 2762576 Alfuy virus Virus 44017 baltica Pseudomonas Bacteria 2843610 Alkhumra Virus 172148 bananamidigenes hemorrhagic fever virus Pseudomonas Bacteria Allpahuayo Virus 144752 bathycetes mammarenavirus Pseudomonas Bacteria 226910 Almeirim virus Virus batumici Pseudomonas Bacteria 556533 Almendravirus Virus 1972683 benzenivorans arboretum Pseudomonas Bacteria 2681983 Almendravirus Virus 1972685 bijieensis cootbay Pseudomonas Bacteria 254015 Almpiwar virus Virus 318843 blatchfordae Pseudomonas Bacteria 2044872 Alocasia Virus 4456 bohemica macrorrhizos Pseudomonas Bacteria 289003 Altamira virus Virus borbori Pseudomonas Bacteria 84586 Amapari virus Virus borealis Pseudomonas Bacteria 2842352 Ambe virus Virus 1926500 botevensis Pseudomonas Bacteria 930166 Amga virus Virus 1511732 brassicacearum Pseudomonas Bacteria 2708063 Amur/Soochong Virus brassicae virus Pseudomonas Bacteria 129817 Anadyr virus Virus 1642852 brenneri Pseudomonas Bacteria 2316085 Anajatuba virus Virus 379964 bubulae Pseudomonas Bacteria 2731681 Ananindeua virus Virus 1927813 campi Pseudomonas Bacteria 915099 Andasibe virus Virus canadensis Pseudomonas Bacteria 2859001 Andes Virus 1980456 canavaninivorans orthohantavirus Pseudomonas Bacteria 86840 Anhanga virus Virus 904722 cannabina Pseudomonas Bacteria 1495066 Anhembi virus Virus 273355 capeferrum Pseudomonas Bacteria 2810614 Anopheles A virus Virus 35307 capsici Pseudomonas Bacteria 46678 Anopheles B virus Virus 35308 caricapapayae Pseudomonas Bacteria 2487355 Anopheles Virus 2053814 carnis flavivirus Pseudomonas Bacteria 1451454 Anopheles Virus 487311 caspiana gambiae densovirus Pseudomonas Bacteria 2320867 Antequera virus Virus 2748239 cavernae Pseudomonas Bacteria 2320866 Apoi virus Virus 64280 cavernicola Pseudomonas Bacteria 651740 Araguari virus Virus 352236 cedrina Pseudomonas Bacteria 155077 Aransas Bay virus Virus 1428582 cellulosa Pseudomonas Bacteria 1583341 Araraquara Virus Virus 139032 cerasi Pseudomonas Bacteria Bluetongue virus Virus 40051 chaetocerotis Pseudomonas Bacteria 489632 Bobaya virus Virus 2818228 chengduensis Pseudomonas Bacteria 203192 Bobia virus Virus chloritidismutans Pseudomonas Bacteria 587753 Boraceia virus Virus chlororaphis Pseudomonas Bacteria 36746 Borna disease Virus 12455 cichorii virus Pseudomonas Bacteria 53408 Botambi virus Virus citronellolis Pseudomonas Bacteria 416340 Boteke virus Virus 864698 clemancea Pseudomonas Bacteria Bouboui virus Virus 64295 coenobios Pseudomonas Bacteria 1605838 Bourbon virus Virus 1618189 coleopterorum Pseudomonas Bacteria 658457 Bovine ephemeral Virus 11303 composti fever virus Pseudomonas Bacteria 200452 Bovine Herpes Virus congelans Virus 1 Pseudomonas Bacteria 53409 Bovine leukemia Virus 11901 coronafaciens virus Pseudomonas Bacteria 47879 Bovine Virus 11246 corrugata orthopneumovirus Pseudomonas Bacteria 168469 Bovine viral Virus 11099 costantinii diarrhea virus 1 Pseudomonas Bacteria 157783 Bowe virus Virus 1400425 cremoricolorata Pseudomonas Bacteria 2724178 Bozo virus Virus 273349 cremoris Pseudomonas Bacteria 2697028 Cumuto virus Virus 1457166 crudilactis Pseudomonas Bacteria 543360 Cupixi Virus 208899 cuatrocienegasensis mammarenavirus Pseudomonas Bacteria 2781239 Curionopolis virus Virus 490110 cyclaminis Pseudomonas Bacteria 2487519 Cyprinid Virus 180230 daroniae herpesvirus 3 Pseudomonas Bacteria 882211 Czech Aedes Virus deceptionensis vexans flavivirus virus Pseudomonas Bacteria 1876757 D′ Aguilar virus Virus defluvii Pseudomonas Bacteria 366289 Dabakala virus Virus delhiensis Pseudomonas Bacteria 43306 Dabieshan virus Virus 1167310 denitrificans Pseudomonas Bacteria Dak Nong virus Virus 1238455 diazotrophicus Pseudomonas Bacteria 135830 Dakar bat virus Virus 64282 diterpeniphila Pseudomonas Bacteria 1163398 Dandenong virus Virus 483046 donghuensis Pseudomonas Bacteria 2487520 Dashli virus Virus 1764087 dryadis Pseudomonas Bacteria 459528 Deer tick virus Virus 58535 duriflava Pseudomonas Bacteria 2006980 Dengue virus Virus 12637 edaphica Pseudomonas Bacteria 2842353 Dengue virus 1 Virus ekonensis virus Pseudomonas Bacteria 179878 Cumuto virus Virus 1457166 elodea Pseudomonas Bacteria 1563157 Cupixi Virus 208899 endophytica mammarenavirus Pseudomonas Bacteria 312306 Curionopolis virus Virus 490110 entomophila Pseudomonas Bacteria 2599595 Lymphocytic Virus 11623 eucalypticola choriomeningitis mammarenavirus Pseudomonas Bacteria Lyssavirus aravan Virus 211977 excibis Pseudomonas Bacteria 359110 Lyssavirus Virus 90961 extremaustralis australis Pseudomonas Bacteria 169669 Lyssavirus lagos Virus 38766 extremorientalis Pseudomonas Bacteria 2842355 Lyssavirus spp. Virus 11286 fakonensis Pseudomonas Bacteria 2841207 Lyssavirus bokeloh Virus 1072176 farris Pseudomonas Bacteria 2745492 Lyssavirus caucasicus Virus 249584 farsensis Pseudomonas Bacteria 53410 Lyssavirus duvenhage Virus 38767 ficuserectae Pseudomonas Bacteria 1674920 Lyssavirus irkut Virus 249583 fildesensis Pseudomonas Bacteria 29435 Lyssavirus khujand Virus 237716 flavescens Pseudomonas Bacteria 706570 Lyssavirus mokola Virus 12538 flexibilis Pseudomonas Bacteria 1958950 Lyssavirus rabies Virus 11292 floridensis Pseudomonas Bacteria 294 Lyssavirus shimoni Virus 746543 fluorescens Pseudomonas Bacteria 1793966 Marisma mosquito Virus 1105173 fluvialis virus Pseudomonas Bacteria 2762593 Marituba virus Virus 292278 foliumensis Pseudomonas Bacteria 296 Marondera virus Virus 108092 fragi Pseudomonas Bacteria 104087 Marrakai virus Virus 108088 frederiksbergensis Pseudomonas Bacteria 200453 Massila virus Virus fulgida Pseudomonas Bacteria 47880 Matariya virus Virus 1272948 fulva Pseudomonas Bacteria 1149133 Matruh virus Virus 1678229 furukawaii Pseudomonas Bacteria 50340 Matucare virus Virus 908873 fuscovaginae Pseudomonas Bacteria 1653853 Mayaro virus Virus 59301 gelidicola Pseudomonas Bacteria 78544 Mboke virus Virus 273342 gessardii Pseudomonas Bacteria 117681 Mburo virus Virus 2035534 gingeri Pseudomonas Bacteria 1577705 Meaban virus Virus 35279 glareae Pseudomonas Bacteria 1785145 Medjerda Valley Virus 1775957 glycinae virus Pseudomonas Bacteria 2774461 Melao virus Virus 35515 gozinkensis Pseudomonas Bacteria 158627 Meno virus Virus graminis Pseudomonas Bacteria 1421430 Mercadeo virus Virus 1708574 granadensis Pseudomonas Bacteria 1628277 Semliki Forest Virus 11033 gregormendelii virus Pseudomonas Bacteria 129847 Sena Madureira Virus 1272957 grimontii virus aPseudomonas Bacteria 1245526 Seoul virus Virus 1980490 guangdongensis Pseudomonas Bacteria 1288410 Sepik virus Virus 44026 guariconensis Pseudomonas Bacteria 310348 Serra Do Navio Virus 45768 guezennei virus Pseudomonas Bacteria 1198456 Serra Norte virus Virus 1000649 guguanensis Pseudomonas Bacteria 425504 Severe fever with Virus 1003835 guineae thrombocytopenia syndrome virus Pseudomonas Bacteria 2759165 Shamonda virus Virus 159150 guryensis Pseudomonas Bacteria 2600065 Shark River virus Virus 2303490 haemolytica Pseudomonas Bacteria 53411 Shiant Island virus Virus halodenitrificans Pseudomonas Bacteria 28258 Shokwe virus Virus 273359 halodurans Pseudomonas Bacteria Shuni virus Virus 159148 halosaccharolytica Pseudomonas Bacteria Silverwater virus Virus 1564099 halosensibilis Pseudomonas Bacteria 2745504 Simbu Virus 35306 hamedanensis orthobunyavirus Pseudomonas Bacteria 251654 Sin Nombre virus Virus 1980491 helianthi Pseudomonas Bacteria 1608996 Sindbis virus Virus 11034 helleri Pseudomonas Bacteria 1471381 Sixgun City virus Virus helmanticensis Pseudomonas Bacteria 2213017 Skinner Tank virus Virus 481886 huaxiensis Pseudomonas Bacteria 1247546 Snowshoe hare Virus 11580 hunanensis virus Pseudomonas Bacteria 2707027 Sokoluk virus Virus 64317 hutmensis Pseudomonas Bacteria 297 Soldado virus Virus 426791 hydrogenothermo phila Pseudomonas Bacteria 39439 Solwezi virus Virus hydrogenovora Pseudomonas Bacteria 2493633 Somone virus Virus hydrolytica Pseudomonas Bacteria 137658 Sororoca virus Virus 273354 indica Pseudomonas Bacteria 404407 Souris virus Virus 2010246 indoloxydans Pseudomonas Bacteria 2078786 South Bay virus Virus 1526514 inefficax Pseudomonas Bacteria 2745503 South River virus Virus 45769 iranensis Pseudomonas Bacteria 2710587 Spanish Culex Virus iridis flavivirus virus Pseudomonas Bacteria 2684212 Spanish Virus izuensis Ochlerotatus flavivirus virus Pseudomonas Bacteria 256466 Spondweni virus Virus 64318 japonica Pseudomonas Bacteria 77298 Sprivirus cyprinus Virus 696863 jessenii Pseudomonas Bacteria Sripur virus Virus 1620897 jinanensis Pseudomonas Bacteria 198616 St. Abbs Head Virus jinjuensis virus Pseudomonas Bacteria 2666183 St. Croix River Virus juntendi virus Pseudomonas Bacteria 2293832 St. Louis Virus 11080 kairouanensis encephalitis virus Pseudomonas Bacteria 1055468 Stanfield virus Virus karstica Pseudomonas Bacteria 2745482 Stratford virus Virus 44027 kermanshahensis

TABLE 6 Exemplary list of viruses NCBI NCBI NCBI Taxonomy Taxonomy Taxonomy Name ID Name ID Name ID Aalivirus A 2169685 Enterovirus A 138948 Pseudomonas 462590 virus Yua Aarhusvirus 2732762 Enterovirus B 138949 Pseudoplusia dagda includens virus Aarhusvirus 2732763 Enterovirus C 138950 Pseudotevenvirus 329381 katbat RB16 Aarhusvirus 2732764 Enterovirus D 138951 Pseudotevenvirus 115991 luksen RB43 Aarhusvirus 2732765 Enterovirus E 12064 Psimunavirus 2734265 mysterion psiM2 Abaca bunchy 438782 Enterovirus F 1330520 Psipapillomavirus 1177762 top virus 1 Abatino 2734574 Enterovirus G 106966 Psipapillomavirus 2170170 macacapox 2 virus Abbeymikolo 2734213 Enterovirus H 310907 Psipapillomavirus 2170171 nvirus 3 abbeymikolon Abouovirus 1984774 Enterovirus I 2040663 Psittacid 50294 abouo alphaherpesvirus 1 Abouovirus 1984775 Enterovirus J 1330521 Psittacine 2003673 davies atadenovirus A Abutilon 1926117 Enterovirus K 2169884 Psittacine 2169709 golden mosaic aviadenovirus B virus Abutilon 932071 Enterovirus L 2169885 Psittacine 2734577 mosaic aviadenovirus C Bolivia virus Abutilon 1046572 Entnonagintavirus 2734061 Psittacinepox 2169712 mosaic Brazil ENT90 virus virus Abutilon 10815 Entoleuca 2734428 Pteridovirus 2734351 mosaic virus entovirus filicis Abutilon 169102 Enytus Pteridovirus 2734352 yellows virus montanus maydis ichnovirus Acadevirus 2733576 Ephemerovirus 1972589 Pteropodid 2560693 PM116 adelaide alphaherpesvirus 1 Acadevirus 2733577 Ephemerovirus 1972594 Pteropox virus 1873698 Pm5460 berrimah Acadevirus 2733574 Ephemerovirus 1972593 Pteropus 1985395 PM85 febris associated gemycircularvirus 1 Acadevirus 2733575 Ephemerovirus 1972595 Pteropus 1985404 PM93 kimberley associated gemycircularvirus 10 Acadianvirus 1982901 Ephemerovirus 1972596 Ptyasnivirus 1 2734501 acadian koolpinyah Acadianvirus 1982902 Ephemeroviru 1972587 Pukovnikvirus 540068 baee kotonkan pukovnik Acadianvirus 1982903 Ephemeroviru 1972592 Pulverervirus 2170091 reprobate obodhiang PFR1 Acanthamoeba 212035 Ephemerovirus 1972597 Puma lentivirus 12804 polyphaga yata mimivirus Acanthocystis 322019 Epichloe 382962 Pumpkin 2518373 turfacea festucae virus polerovirus chlorella virus 1 1 Acara 2170053 Epinotia 166056 Pumpkin yellow 1410062 orthobunyavirus aporema mosaic virus granulovirus Achimota 2560259 Epiphyas 70600 Punavirus P1 10678 pararubulavirus postvittana 1 nucleopolyhed rovirus Achimota 2560260 Epirus cherry 544686 Punavirus RCS47 2560452 pararubulavirus virus 2 Achromobacter 2169962 Epizootic 100217 Punavirus SJ46 2560732 virus Axp3 haematopoietic necrosis virus Acidianus 437444 Epizootic 40054 Punique 2734468 bottle-shaped hemorrhagic phlebovirus virus disease virus Acidianus 300186 Eponavirus 2734105 Punta Toro 1933186 filamentous epona phlebovirus virus 2 Acidianus 346881 Epseptimavirus 1982565 Puumala 1980486 filamentous 118970sal2 orthohantavirus virus 3 Acidianus 346882 Epseptimavirus 491003 Pyrobaculum 1805492 filamentous EPS7 filamentous virus virus 6 1 Acidianus 346883 Epseptimavirus 2732021 Pyrobaculum 270161 filamentous ev123 spherical virus virus 7 Acidianus 346884 Epseptimavirus 2732022 Qadamvirus 2733953 filamentous SB28 virus 8 Acidianus 512792 Epseptimavirus 2732023 Qalyub 1980527 filamentous LVR16A orthonairovirus virus 9 Acidianus 309181 Epseptimavirus 2732019 Qingdaovirus J21 2734135 rod-shaped mar003J3 virus 1 Acidianus 693629 Epseptimavirus 2732024 Qingling 2560694 spindle- S113 orthophasmavirus shaped virus 1 Acidianus 315953 Epseptimavirus 2732025 Quail pea mosaic two-tailed S114 virus virus Acinetobacter 279006 Epseptimavirus 2732026 Quailpox virus 400570 virus 133 S116 Acintetobacter Epseptimavirus 2732027 Quaranjavirus 688437 virus B2 S124 johnstonense Acintetobacter Epseptimavirus 2732028 Quaranjavirus 688436 virus B5 S126 quaranfilense Acionnavirus 2734078 Epseptimavirus 2732029 Qubevirus durum 39803 monteraybay S132 Acipenserid 2871198 Epseptimavirus 2732030 Qubevirus 39804 herpesvirus 2 S133 faecium Aconitum 101764 Epseptimavirus 2732031 Quezon 2501382 latent virus S147 mobatvirus Acrobasis Epseptimavirus 2732020 Quhwahvirus 2283289 zelleri saus 132 kaihaidragon entomopoxvirus Actinidia seed 2560282 Epseptimavirus 2732032 Quhwahvirus 2201441 borne latent seafire ouhwah virus Actinidia 2024724 Epseptimavirus 2732033 Quhwahvirus 2182400 virus 1 SH9 paschalis Actinidia 1112769 Epseptimavirus 2732034 Rabbit associated 1985420 virus A STG2 gemykroznavirus 1 Actinidia 1112770 Epseptimavirus 1540099 Rabbit fibroma 10271 virus B stitch virus Actinidia 1331744 Epseptimavirus 2732035 Rabbit 11976 virus X Sw2 hemorrhagic disease virus Acute bee 92444 Epsilonarterivirus 2501964 Rabovirus A 1603962 paralysis virus hemcep Adana 2734433 Epsilonarterivirus 2501965 Rabovirus B 2560695 phlebovirus safriver Adeno- 1511891 Epsilonarterivirus 2501966 Rabovirus C 2560696 associated zamalb dependoparvo virus A Adeno- 1511892 Epsilonpapillo 40537 Rabovirus D 2560697 associated mavirus 1 dependoparvo virus B Adoxophyes 1993630 Epsilonpapillo 2169886 Raccoonpox 10256 honmai mavirus 2 virus entomopoxvirus Adoxophyes 224399 Epsilonpolyo 1891754 Radish leaf curl 435646 honmai mavirus bovis virus nucleopolyhed rovirus Adoxophyes 170617 Eptesipox 1329402 Radish mosaic 328061 orana virus virus granulovirus Aedes aegypti Equid 10326 Radish yellow 319460 entomopoxvirus alphaherpesvirus edge virus 1 Aedes aegypti Equid 80341 Rafivirus A Mosqcopia alphaherpesvirus virus 3 Aedes 341721 Equid 10331 Rafivirus B 2560699 pseudoscutella alphaherpesvirus ris reovirus 4 Aegirvirus 2733888 Equid 39637 Rafivirus C SCBP42 alphaherpesvirus 8 Aeonium 1962503 Equid 55744 Raleighvirus 2734266 ringspot virus alphaherpesvirus darolandstone 9 Aeromonas Equid 12657 Raleighvirus 2734267 virus 43 gammaherpes raleigh virus 2 Aeropyrum 1157339 Equid 10371 Ramie mosaic 1874886 coil-shaped gammaherpes Yunnan virus virus virus 5 Aeropyrum 700542 Equid 291612 Ranid 85655 pernix gammaherpes herpesvirus 1 bacilliform virus 7 virus 1 Aeropyrum 1032474 Equine 1985379 Ranid 389214 pernix ovoid associated herpesvirus 2 virus 1 gemycircularvirus 1 Aerosvirus 2733365 Equine 201490 Ranid 1987509 AS7 encephalosis herpesvirus 3 virus Aerosvirus 2733364 Equine foamy 109270 Ranunculus leaf 341110 av25AhydR2PP virus distortion virus Aerosvirus 2733366 Equine 11665 Ranunculus mild 341111 ZPAH7 infectious mosaic virus anemia virus Affertcholera 141904 Equine 129954 Ranunculus 341112 mvirus mastadenovirus mosaic virus CTXphi A African 2560285 Equine 129955 Raptor 691961 cassava mastadenovirus siadenovirus A mosaic B Burkina Faso virus African 10817 Equine 2723956 Raspberry bushy 12451 cassava picobirnavirus dwarf virus mosaic virus African 2056161 Equine rhinitis 47000 Raspberry leaf 326941 eggplant A virus mottle virus mosaic virus African horse 40050 Equine 329862 Raspberry 12809 sickness virus torovirus ringspot virus African oil 185218 Eracentumvirus 1985737 Rat associated 1985405 palm ringspot era103 gemycircularvirus virus 1 African swine 10497 Eracentumvirus 2733579 Rat associated 2170126 fever virus S2 porprismacovirus 1 Agaricus 2734345 Eragrostis 638358 Rattail cactus 1123754 bisporus curvula streak necrosis- alphaendornavirus virus associated virus 1 Agaricus Eragrostis 1030595 Rattus norvegicus 1679933 bisporus virus minor streak polyomavirus 1 4 virus Agatevirus 1910935 Eragrostis 496807 Rauchvirus BPP1 194699 agate streak virus Agatevirus 1910936 Erbovirus A 312185 Raven circovirus 345250 bobb Agatevirus 1910937 Erectites 390443 Ravinvirus N15 40631 Bp8pC yellow mosaic virus Ageratum 1260769 Eriborus Recovirus A 2560702 enation terebrans alphasatellite ichnovirus Ageratum 188333 Erinnyis ello 307444 Red clover enation virus granulovirus associated luteovirus Ageratum 1386090 Eriocheir 273810 Red clover 1323524 latent virus sinensis cryptic virus 2 reovirus Ageratum leaf 912035 Ermolevavirus 2733903 Red clover mottle 12262 curl Buea PGT2 virus betasatellite Ageratum leaf 635076 Ermolevavirus 2733904 Red clover 12267 curl PhiKT necrotic mosaic Cameroon virus betasatellite Ageratum leaf 2182585 Erskinevirus 2169882 Red clover vein 590403 curl Sichuan asesino mosaic virus virus Ageratum leaf 333293 Erskinevirus 2169883 Red deerpox curl virus EaH2 virus Ageratum 169687 Erysimum 12152 Redspotted 43763 yellow leaf latent virus grouper nervous curl necrosis virus betasatellite Ageratum 187850 Feline 1987742 Reginaelenavirus 2734071 yellow vein associated rv3LV2017 alphasatellite cyclovirus 1 Ageratum 185750 Feline 11978 Rehmannia 425279 yellow vein calicivirus mosaic virus betasatellite Ageratum 1454227 Feline foamy 53182 Rehmannia virus 2316740 yellow vein virus 1 China alphasatellite Ageratum 437063 Feline 11673 Reptilian 122203 yellow vein immunodeficiency ferlavirus Hualian virus virus Ageratum 1407058 Feline 11768 Reptilian 226613 yellow vein leukemia virus orthoreovirus India alphasatellite Ageratum 2010316 Feline 1170234 Rerduovirus 1982376 yellow vein morbillivirus RER2 India betasatellite Ageratum 915293 Felipivirus A Rerduovirus 1109716 yellow vein RGL3 Singapore alphasatellite Ageratum 2010317 Felixounavirus 2560439 Restivirus RSS1 2011075 yellow vein Alf5 Sri Lanka betasatellite Ageratum 222079 Felixounavirus 1965378 Reston ebolavirus 186539 yellow vein AYO145A Sri Lanka virus Ageratum 44560 Felixounavirus 2560723 Reticuloendotheliosis 11636 yellow vein BPS15Q2 virus virus Aghbyvirus 2733367 Felsduovirus 2734062 Reyvirus rey 1983751 ISAO8 4LV2017 Aglaonema 1512278 Felsduovirus 194701 Rhesus macaque 2170199 bacilliform Fels2 simian foamy virus virus Agricanvirus 1984777 Felsduovirus 2734063 Rhinolophus 2004965 deimos RE2010 associated gemykibivirus 1 Agricanvirus 2560433 Felsduovirus 2734062 Rhinolophus 2004966 desertfox 4LV2017 associated gemykibivirus 2 Agricanvirus 1984778 Felsduovirus 194701 Rhinolophus bat 693998 Ea3570 Fels2 coronavirus HKU2 Agricanvirus 1984779 Fernvirus 1921560 Rhinolophus 2501926 ray shelly ferrumequinum alphacoronavirus HuB-2013 Agricanvirus 1984780 Fernvirus 1921561 Rhinovirus A 147711 simmy50 sitara Agricanvirus 1984781 Festuca leaf Rhinovirus B 147712 specialG streak cytorhabdovirus Agropyron 41763 Fibralongavirus 2734233 Rhinovirus C 463676 mosaic virus fv2638A Agrotis 208013 Fibralongavirus 2734234 Rhizidiomyces ipsilon QT1 virus multiple nucleopolyhed rovirus Agrotis 10464 Fibrovirus fs1 70203 Rhizoctonia 1408133 segetum cerealis granulovirus alphaendornavirus 1 Agrotis 1962501 Fibrovirus 1977140 Rhizoctonia 2560704 segetum VGJ magoulivirus 1 nucleopolyhed rovirus A Agrotis 1580580 Ficleduovirus 2560473 Sabo 2560716 segetum FCL2 orthobunyavirus nucleopolyhed rovirus B Agtrevirus 1987994 Ficleduovirus 2560474 Saboya virus 64284 AG3 FCV1 Agtrevirus 2169690 Fig badnavirus 1034096 Sacbrood virus 89463 SKML39 1 Aguacate 2734434 Fig cryptic 882768 Saccharomyces 186772 phlebovirus virus 20S RNA narnavirus Ahlum Figulus Saccharum streak 683179 waterborne sublaevis virus virus entomopoxvirus Ahphunavirus 2733368 Figwort 10649 Saclayvirus 2734138 Ahp1 mosaic virus Aci011 Ahphunavirus 2733369 Fiji disease 77698 Saclayvirus 2734139 CF7 virus Aci022 Ahtivirus 2734079 Finch 400122 Saclayvirus 2734137 sagseatwo circovirus Aci05 Aichivirus A 72149 Finkel-Biskis- 353765 Saetivirus fs2 1977306 Jinkins murine sarcoma virus Aichivirus B 194965 Finnlakevirus 2734591 Saetivirus VFJ 1977307 FLip Aichivirus C 1298633 Fionnbharthvirus 2955891 Saffron latent 2070152 fionnbharth virus Aichivirus D 1897731 Fipivirus A Saguaro cactus 52274 virus Aichivirus E 1986958 Fipvunavirus 2560476 Saguinine 2169901 Fpv4 gammaherpesvirus 1 Aichivirus F 1986959 Firehammervirus 1190451 Saikungvirus 2169924 CP21 HK633 Ailurivirus A 2560287 Firehammervirus 722417 Saikungvirus 2169925 CP220 HK75 Aino 2560289 Firehammervirus 722418 Saimiri sciureus 1236410 orthobunyavirus CPt10 polyomavirus 1 Air potato 2560290 Fischettivirus 230871 Saimiriine 10353 ampelovirus 1 C1 alphaherpesvirus 1 Akabane 1933178 Fishburnevirus 1983737 Saimiriir 1535247 orthobunyavirus brusacoram betaherpesvirus 4 Akhmeta virus 2200830 Flamingopox 503979 Saimiriine 10381 virus gammaherpesvirus 2 Alajuela 1933181 Flammulina 568090 Saint Floris orthobunyavirus velutipes phlebovirus browning virus Alasvirus 2501934 Flaumdravirus 2560665 Saint Louis 11080 muscae KIL2 encephalitis virus Alcelaphine 35252 Flaumdravirus 2560666 Saint Valerien gammaherpes KIL4 virus virus 1 Alcelaphine 138184 Fletchervirus 1980966 Sakhalin 1980528 gammaherpes CP30A orthonairovirus virus 2 Alcube 2734435 Gaiavirus gaia 1982148 Sakobuvirus A 1659771 phlebovirus Alcyoneusvirus 2560541 Gaillardia 1468172 Sal Vieja virus 64301 K641 latent virus Alcyoneusvirus 2560545 Gairo 1535802 Salacisavirus 2734140 RaK2 mammarenavirus pssm2 Alefpapilloma 2169692 Gajwadongvirus 2733916 Salanga 2734471 virus 1 ECBP5 phlebovirus Alenquer 2734436 Gajwadongvirus 2733917 Salasvirus phi29 10756 phlebovirus PP99 Alexandravirus 2734080 Galaxyvirus 2560298 Salchichonvirus 298338 AD1 abidatro LP65 Alexandravirus 2734081 Galaxyvirus 2560303 Salehabad 1933188 alexandra galaxy phlebovirus Alfalfa Galinsoga 60714 Salem salemvirus 2560718 betanucleorha mosaic virus bdovirus Alfalfa cryptic Gallid 10386 Salivirus A 1330524 virus 1 alphaherpesvirus 1 Alfalfa 1770265 Gamaleyavirus 1920761 Salmo 2749930 enamovirus 1 Sb1 aquaparamyxovirus Alfalfa leaf 1306546 Gambievirus 2501933 Salmon gillpox 2734576 curl virus bolahunense virus Alfalfa mosaic 12321 Gamboa 1933270 Saphexavirus 1982380 virus orthobunyavirus VD13 Alfalfa virus S 1985968 Gammaarterivirus 2499678 Sapporo virus 95342 lacdeh Algerian 515575 Gammanucleo 2748968 Sarcochilus virus 104393 watermelon rhabdovirus Y mosaic virus maydis Allamanda 452758 Gammapapillo 333926 Sashavirus sasha 2734275 leaf curl virus mavirus 1 Allamanda 1317107 Gammapapillo 1175852 Sasquatchvirus 2734143 leaf mottle mavirus 10 Y3 distortion virus Alligatorweed Gammapapillo 1513256 Sasvirus BFK20 2560392 stunting virus mavirus 11 Allium cepa 2058778 Gayfeather 578305 Satsuma dwarf 47416 amalgavirus 1 mild mottle virus virus Allium cepa 2058779 Gecko 2560481 Sauletekiovirus 2734030 amalgavirus 2 reptillovirus AAS23 Allium virus 317027 Gelderlandvirus 2560727 Saumarez Reef 40012 X melville virus Allpahuayo 144752 Gelderlandvirus 1913658 Saundersvirus 2170234 mammarenavirus s16 Tp84 Almendravirurus 1972686 Gelderlandvirus 1913657 Sauropus leaf 1130981 almendras stml198 curl virus Almendravirus 1972683 Gelderlandvirus 2560734 Sawgrhavirus 2734397 arboretum stp4a connecticut Almendravirus 1972684 Gentian 182452 Sawgrhavirus 2734398 balsa mosaic virus longisland Almendravirus 1972687 Gentian ovary 1920772 Sawgrhavirus 2734399 chico ringspot virus minto Almendravirus 1972685 Geotrupes Sawgrhavirus 2734400 cootbay sylvaticus sawgrass entomopoxvirus Almendravirus 2734366 Gequatrovirus 1986034 Scale drop 1697349 menghai G4 disease virus Bat associated 1987731 Gequatrovirus 1910968 Scallion mosaic 157018 cyclovirus 6 ID52 virus Bat associated 1987732 Gequatrovirus 1910969 Scapularis 2734431 cyclovirus 7 talmos ixovirus Bat associated 1987733 Gerygone 1985381 Scapunavirus 2560792 cyclovirus 8 associated scap1 gemycircularvirus 1 Bat associated 1987734 Gerygone 1985382 Scheffersomyces 1300323 cyclovirus 9 associated segobiensis virus gemycircularvirus L 2 Bat 1913643 Harrisina 115813 Schefflera 2169729 coronavirus brillians ringspot virus CDPHE15 granulovirus Bat 1244203 Harrisonvirus 1982221 Schiekvirus 2560422 coronavirus harrison EFDG1 HKU10 Bat Hp- 2501961 Harvey 11807 Schiekvirus 2734044 betacoronavirus murine EFP01 sarcoma virus Zhejiang2013 Bat 1146877 Hautrevirus 1982895 Schiekvirus 2734045 mastadenovirus hau3 EfV12 A Bat 1146874 Havel River 254711 Schistocerca mastadenovirus virus gregaria B entomopoxvirus Bat 2015370 Hawkeyevirus 2169910 Saphexavirus 1982380 mastadenovirus hawkeye VD13 C Bat 2015372 Hazara 1980522 Sophora yellow 2169837 mastadenovirus orthonairovirus stunt D alphasatellite 5 Bat 2015374 Heartland 2747342 Sorex araneus 2734504 mastadenovirus bandavirus coronavirus T14 E Bat 2015375 Hebius Sorex araneus 2560769 mastadenovirus tobanivirus 1 polyomavirus 1 F Bat 2015376 Hedgehog 1965093 Sorex coronatus 2560770 mastadenovirus coronavirus 1 polyomavirus 1 G Bat Hedwigvirus 2560502 Sorex minutus 2560771 mastadenovirus hedwig polyomavirus 1 H Bat Hedyotis 1428190 Sorghum 107804 mastadenovirus uncinella chlorotic spot yellow mosaic virus virus Bat Hedyotis 1428189 Sorghum mosaic 32619 mastadenovirus yellow mosaic virus J betasatellite Batai 2560341 Heilongjiangv 2734110 Sororoca 2560772 orthobunyavirus irus Lb orthobunyavirus Batama 1933177 Helenium 12171 Sortsnevirus 2734190 orthobunyavirus virus S IME279 Batfish 2560342 Helianthus 2184469 Sortsnevirus 2734189 actinovirus annuus sortsne alphaendornavirus Bavaria virus 2560343 Helicobasidium 675833 Sosuga 2560773 mompa pararubulavirus alphaendornavirus 1 Baxtervirus 2169730 Helicobasidium 344866 Soupsvirus soups 1982563 baxterfox mompa partitivirus V70 Baxtervirus 2169731 Helicobasidium 196690 Soupsvirus 2560510 yeezy mompa strosahl totivirus 1-17 Baylorvirus 2734055 Helicoverpa 489830 Soupsvirus wait 2560513 bv1127AP1 armigera granulovirus Baylorvirus 376820 Helicoverpa 51313 Souris 2169997 PHL101 armigera mammarenavirus nucleopolyhed rovirus Bayou 1980459 Helicoverpa 37206 Sourvirus sour 2560509 orthohantavirus armigera stunt virus Bcepfunavirus 417280 Heliothis 10290 South African 63723 bcepF1 armigera cassava mosaic entomopoxvirus virus Bcepmuvirus 264729 Heliothis 113366 Southern bean 12139 bcepMu virescens mosaic virus ascovirus 3a Bcepmuvirus 431894 Heliothis zea 29250 Southern cowpea 196398 E255 nudivirus mosaic virus Bdellomicrovirus 1986027 Helleborus 592207 Southern 1159195 MH2K mosaic virus elephant seal virus Bdellovibrio Helleborus net 592206 Southern rice 519497 virus MAC1 necrosis virus black-streaked dwarf virus Beak and 77856 Helminthosporium 2560520 Southern tomato 591166 feather disease victoriae virus virus virus 145S Bean calico 31602 Helminthosporium 45237 Sowbane mosaic 378833 mosaic virus victoriae virus virus 190S Bean chlorosis 1227354 Helsettvirus 2733626 Soybean 1985413 virus fPS53 associated gemycircularvirus 1 Bean common 43240 Helsettvirus 2733628 Sophora yellow 2169837 mosaic fPS54ocr stunt necrosis virus alphasatellite 5 Bean common 12196 Helsettvirus 2733627 Sorex araneus 2734504 mosaic virus fPS59 coronavirus T14 Bean dwarf 10838 Helsettvirus 2733625 Sorex araneus 2560769 mosaic virus fPS9 polyomavirus 1 Bean golden 10839 Helsingorvirus 1918193 Sorex coronatus 2560770 mosaic virus Cba121 polyomavirus 1 Bean golden 220340 Helsingorvirus 1918194 Sorex minutus 2560771 yellow mosaic Cba171 polyomavirus 1 virus Bean leaf 2004460 Jujube 2020956 Sorghum 107804 crumple virus mosaic- chlorotic spot associated virus virus Bean leafroll 12041 Jun 2560536 Sorghum mosaic 32619 virus jeilongvirus virus Bean mild Juncopox Sororoca 2560772 mosaic virus virus orthobunyavirus Bean necrotic 2560344 Jutiapa virus 64299 Sortsnevirus 2734190 mosaic IME279 orthotospovirus Bean pod 12260 Jwalphavirus 2169963 Switchgrass 2049938 mottle virus jwalpha mosaic- associated virus Bean rugose 128790 Kabuto 2747382 Symapivirus A mosaic virus mountain uukuvirus Bean white 2169732 Kadam virus 64310 Synechococcus 2734100 chlorosis virus SRIM12-08 mosaic virus Bean yellow 267970 Kadipiro virus 104580 Synedrella leaf 1544378 disorder virus curl alphasatellite Bean yellow 714310 Kaeng Khoi 1933275 Synedrella 1914900 mosaic orthobunyavirus yellow vein Mexico virus clearing virus Bean yellow 12197 Kafavirus 2733923 Synetaeris mosaic virus SWcelC56 tenuifemur ichnovirus Bear Canyon 192848 Kafunavirus 1982588 Syngnathid 2734305 mammarenavirus KF1 ichthamaparvovirus 1 Beauveria 1740646 Kagunavirus 2560464 Synodus 2749934 bassiana golestan synodonvirus polymycovirus 1 Beauveria 1685109 Kagunavirus 1911008 Tabernariusvirus 2560691 bassiana K1G tabernarius victorivirus 1 Bebaru virus 59305 Kagunavirus 1911010 Tacaiuma 611707 K1H orthobunyavirus Beecentumtre 10778 Kagunavirus 1911007 Tacaribe 11631 virus B103 Klind1 mammarenavirus Beet black 196375 Kagunavirus 1911009 Tacheng 2734606 scorch virus Klind2 uukuvirus Beet chlorosis 131082 Kagunavirus 2734197 Tahyna 2560796 virus RP180 orthobunyavirus Beet cryptic 509923 Merremia 77813 Tangaroavirus 2733962 virus 1 mosaic virus tv951510a Beet cryptic 912029 Mesta yellow 1705093 Tankvirus tank 1982567 virus 2 vein mosaic alphasatellite Beet cryptic 29257 Mesta yellow 508748 Tapara 2734474 virus 3 vein mosaic phlebovirus Bahraich virus Beet curly top 391228 Metamorphoo 2734253 Tapirape 2560798 Iran virus virus fireman pacuvirus Beet curly top 10840 Metamorphoo 2734254 Tapwovirus cesti 2509383 virus virus metamorphoo Beet mild 156690 Metamorphoo 2734255 Taranisvirus 2734146 yellowing virus robsfeet taranis virus Beet mosaic 114921 Metrivirus 2560269 Taro bacilliform 1634914 virus ME3 CH virus Beet necrotic 31721 Mguuvirus 2733593 Taro bacilliform 178354 yellow vein JG068 virus virus Beet 72750 Microbacterium Tarumizu 2734340 pseudoyellows virus coltivirus virus MuffinTheCat [2] Beet ringspot 191547 Microcystis 340435 Tataguine 2560799 virus virus Ma- orthobunyavirus LMM01 Beet soil- 76343 Microhyla Taterapox virus 28871 borne mosaic letovirus 1 virus Beet soil- 46436 Micromonas 338781 Taupapillomavirus 1176148 borne virus pusilla 1 reovirus Beet virus Q 71972 Micromonas 373996 Taupapillomavirus 1513274 pusilla virus 2 SP1 Beet western 12042 Microplitis Taupapillomavirus 1961786 yellows virus croceipes 3 bracovirus Beet yellow 35290 Microtus 2006148 Taupapillomavirus 2170222 stunt virus arvalis 4 polyomavirus 1 Beet yellows 12161 Mukerjeevirus 2734186 Taura syndrome 142102 virus mv52B1 virus Beetle mivirus Mulberry 1227557 Tawavirus JSF7 2733965 badnavirus 1 Beetrevirus 2560656 Mulberry 1631303 Tea plant 2419939 B3 mosaic dwarf necrotic ring associated blotch virus virus Beetrevirus 2560663 Mulberry 1527441 Tefnutvirus 2734147 JBD67 mosaic leaf siom 18 roll associated virus Beetrevirus 2560664 Mulberry Tegunavirus r1rt 1921705 JD18 ringspot virus Beetrevirus 2560675 Mulberry vein Tegunavirus 1921706 PM105 banding yenmtg1 associated orthotospovirus Beihai Mule deerpox 304399 Tehran 2734475 picobirnavirus virus phlebovirus Beilong 2560345 Mume virus A 2137858 Telfairia golden 2169737 jeilongvirus mosaic virus Bell pepper 354328 Mumps 2560602 Telfairia mosaic 1859135 alphaendornavirus orthorubulavirus virus Bell pepper 368735 Mungbean 2010322 Tellina virus 359995 mottle virus yellow mosaic betasatellite Belladonna 12149 Mukerjeevirus 2734186 Tellina virus 1 321302 mottle virus mv52B1 Bellamyvirus 2734095 Mulberry 1227557 Telosma mosaic 400394 bellamy badnavirus 1 virus Bellavista 2560346 Mulberry 1631303 Tembusu virus 64293 orthobunyavirus mosaic dwarf associated virus Bellflower 1720595 Mycobacterium 1993864 Tensaw 2560800 vein chlorosis virus orthobunyavirus virus Tweety Bellflower 1982660 Mycobacterium 1993860 Tent-making bat 1508712 veinal mottle virus Wee hepatitis B virus virus Beluga whale 694015 Mycobacterium 1993859 Teseptimavirus 2733885 coronavirus virus YpsPG SW1 Wildcat Bendigovirus 2560495 Mycoreovirus 311228 Testudine GMA6 1 orthoreovirus Benedictvirus 1071502 Mycoreovirus 404237 Testudinid 2560801 cuco 2 alphaherpesvirus 3 Benedictvirus 1993876 Mycoreovirus 311229 Tete 35319 tiger 3 orthobunyavirus Benevides 2170054 Mylasvirus 1914020 Tetterwort vein 1712389 orthobunyavirus persius chlorosis virus Bequatrovirus 1984785 Mynahpox 2169711 Teviot 2560803 avesobmore virus pararubulavirus Bequatrovirus 1918005 Myodes Thailand 1980492 B4 coronavirus orthohantavirus 2JL14 Bequatrovirus 1918006 Myodes 2006147 Thalassavirus 2060093 bigbertha glareolus thalassa polyomavirus 1 Bequatrovirus 1918007 Myodes 2560609 Thaumasvirus 2734148 riley jeilongvirus stim4 Bequatrovirus 1918008 Myodes 2560610 Thermoproteus 292639 spock narmovirus tenax spherical virus 1 Bequatrovirus 1918009 Myohalovirus 1980944 Thermoproteus 10479 troll phiH tenax virus 1 Berhavirus 2509379 Noxifervirus 2560671 Thermus virus 1714273 beihaiense noxifer IN93 Berhavirus 2509380 Ntaya virus 64292 Thermus virus 1714272 radialis P23-77 Berhavirus 2509381 Ntepes 2734464 Thetaarterivirus 2501999 sipunculi phlebovirus kafuba Berisnavirus 1 2734518 Nuarterivirus Thetaarterivirus 2502000 guemel mikelba 1 Cacao yellow 12150 Nudaurelia 85652 Thetapapillomavirus 197772 mosaic virus capensis beta 1 virus Cacao yellow 2169726 Nudaurelia 12541 Thetapolyomavirus 1891755 vein banding capensis censtriata virus omega virus Cache Valley 2560364 Nupapillomavirus 334205 Thetapolyomavirus 2218588 orthobunyavirus 1 trebernacchii Cachoeira 2560365 Nyando 1933306 Thetapolyomavirus 2170103 Porteira orthobunyavirus trepennellii orthobunyavirus Cacipacore 64305 Nyavirus 644609 Thetisvirus ssm1 2734149 virus midwayense Cactus mild 229030 Nyavirus 644610 Thiafora 1980529 mottle virus nyamaniniense orthonairovirus Cactus virus 2 Nyavirus 1985708 Thimiri 1819305 sierranevadaense orthobunyavirus Cactus virus X 112227 Nyceiraevirus 2560506 Thin paspalum 1352511 nyceirae asymptomatic virus Cadicivirus A 1330068 Nyctalus 2501928 Thistle mottle velutinus virus alphacoronavirus SC-2013 Cadicivirus B 2560366 Nylanderia 1871153 Thogotovirus 11318 fulva virus 1 dhoriense Caenorhabditis Nymphadoravirus 2170041 Thogotovirus 11569 elegans Cer1 kita thogotoense virus Caenorhabditis Nymphadoravirus 2560507 Thomixvirus 2560804 elegans OH3 Cer 13 virus nymphadora Caeruleovirus 1985175 Nymphadoravirus 2170042 Thornevirus 2560336 Bc431 zirinka SP15 Caeruleovirus 1985176 Oat blue 56879 Thosea asigna 83810 Bcp1 dwarf virus virus Caeruleovirus 1985177 Oat chlorotic 146762 Thottopalayam 2501370 BCP82 stunt virus thottimvirus Caeruleovirus 1985178 Oat dwarf 497863 Thunberg 299200 BM15 virus fritillary mosaic virus Caeruleovirus 1985179 Oat golden 45103 Thysanoplusia 101850 deepblue stripe virus orichalcea nucleopolyhedro virus Caeruleovirus 1985180 Oxbow 1980484 Tiamatvirus 268748 JBP901 orthohantavirus PSSP7 Cafeteria 1513235 Oxyplax 2083176 Tibetan frog 2169919 roenbergensis ochracea hepatitis B virus virus nucleopoly- hedrovirus Cafeteriavirus 1932923 Paadamvirus 2733939 Tibrovirus 1987018 -dependent RHEph01 alphaekpoma mavirus Caimito 2734421 Pacific coast Tibrovirus 2170224 pacuvirus uukuvirus beatrice Cajanus cajan Pacui 2560617 Tibrovirus 1987019 Panzee virus pacuvirus betaekpoma Caladenia 1198147 Paenibacillus Tibrovirus 1972586 virus A virus Willow coastal Calanthe mild 73840 Pagavirus 2733940 Tibrovirus congo 1987017 mosaic virus S05C849 Cali 2169993 Pagevirus 1921185 Tibrovirus 1987013 mammarenavirus page sweetwater Calibrachoa 204928 Pagevirus 1921186 Tibrovirus 1972584 mottle virus palmer tibrogargan California 1933264 Pagevirus 1921187 Tick associated 2560805 encephalitis pascal circovirus 1 orthobunyavirus California 2170175 Pagevirus 1921188 Tick associated 2560806 reptarenavirus pony circovirus 2 Caligid Pagevirus 1921189 Tick-borne 11084 hexartovirus pookie encephalitis virus Caligrhavirus 2560367 Pagoda yellow 1505530 Tico phebovirus 2734476 caligus mosaic associated virus Caligrhavirus 2560551 Paguronivirus 2508237 Tidunavirus 2560834 lepeophtheirus 1 pTD1 Caligrhavirus 2560736 Pahexavirus 1982252 Tidunavirus 2560833 salmonlouse ATCC29399BC VP4B Calla lily 2560368 Pahexavirus 1982303 Tiger puffer 43764 chlorotic spot pirate nervous necrosis orthotospovirus virus Calla lily 243560 Pahexavirus 1982304 Tigray 2560807 latent virus procrass 1 orthohantavirus Callistephus 1886606 Pahexavirus 1982305 Tigrvirus E122 431892 mottle virus SKKY Callitrichine 106331 Pahexavirus 1982306 Tigrvirus E202 431893 gammaherpes solid virus 3 Calopogonium Pahexavirus 1982307 Tobacco leaf curl 439423 yellow vein stormborn Comoros virus virus Camel 2169876 Pahexavirus 1982308 Tobacco leaf curl 336987 associated wiZZO Cuba virus drosmacovirus 1 Camel 2169877 Pahsextavirus 2733975 Tobacco leaf curl 2528965 associated pAh6C Dominican drosmacovirus Republic virus 2 Camel 2170105 Pairvirus 2733941 Tobacco leaf curl 2010326 associated Lo5R7ANS Japan porprismacovirus betasatellite 1 Camel 2170106 Pakpunavirus 1921409 Tobacco leaf curl 2010327 associated CAb02 Patna porprismacovirus betasatellite 2 Camel 2170107 Pahexavirus 1982303 Tobacco leaf curl 905054 associated pirate Pusa virus porprismacovirus 3 Camel 2170108 Pahexavirus 1982304 Tobacco leaf curl 409287 associated procrass 1 Thailand virus porprismacovirus 4 Camelpox 28873 Pahexavirus 1982305 Tobacco leaf curl 211866 virus SKKY Yunnan virus Campana 2734442 Pea necrotic 753670 Tobacco leaf curl 223337 phlebovirus yellow dwarf Zimbabwe virus virus Campoletis Pea seed- 12208 Tobacco leaf 196691 aprilis borne mosaic rugose virus ichnovirus virus Campoletis Pea stem 199361 Veracruzvirus 1032892 flavicincta necrosis virus heldan ichnovirus Camptochiron Pea streak 157777 Veracruzvirus 2003502 omus tentans virus rockstar entomopoxvirus Campylobacter 1006972 Pea yellow 1436892 Verbena latent 134374 virus IBB35 stunt virus virus Camvirus 1982882 Peach 471498 Verbena virus Y 515446 amela chlorotic mottle virus Camvirus 1982883 Peach latent 12894 Vernonia crinkle 1925153 CAM mosaic viroid virus Canary 142661 Peach 2169999 Vernonia yellow 666635 circovirus marafivirus D vein betasatellite Canarypox 44088 Peach mosaic 183585 Vernonia yellow 2169908 virus virus vein Fujian alphasatellite Candida Peach rosette 65068 Vernonia yellow 2050589 albicans Tca2 mosaic virus vein Fujian virus betasatellite Candida Peanut 35593 Vernonia yellow 1001341 albicans Tca5 chlorotic vein Fujian virus virus streak virus Candiru 1933182 Peanut clump 28355 Vernonia yellow 367061 phlebovirus virus vein virus Canid 170325 Peanut yellow Versovirus 2011076 alphaherpesvirus mosaic virus VfO3K6 1 Canine 1985425 Pear blister 12783 Verticillium 759389 associated canker viroid dahliae gemygorvirus chrysovirus 1 1 Canine 1194757 Peaton 2560627 Vesicular 35612 circovirus orthobunyavirus exanthema of swine virus Canine 10537 Peatvirus 2560629 Vesiculovirus 1972579 mastadenovirus peat2 alagoas A Canine 11232 Pecan mosaic- 1856031 Vesiculovirus 1972567 morbillivirus associated bogdanovac virus Canna yellow 2560371 Pecentumvirus 40523 Whitefly- 2169744 mottle A511 associated associated begomovirus 7 virus Canna yellow 419782 Penicillum 2734569 White-tufted-ear 2170205 mottle virus brevicompactum marmoset simian polymycovirus foamy virus 1 Canna yellow 433462 Pennisetum 221262 Whitewater 46919 streak virus mosaic virus Arroyo mammarenavirus Cannabis 1115692 Pepino mosaic Wifcevirus 2734154 cryptic virus virus [3] ECML117 Cano 1980463 Pepo aphid- 1462681 Wifcevirus 2734155 Delgadito borne yellows FEC19 orthohantavirus virus Canoevirus 2734056 Pepper chat 574040 Wifcevirus WFC 2734156 canoe fruit viroid Cao Bang 1980464 Pepper 2734493 Wifcevirus WFH 2734157 orthohantavirus chlorotic spot orthotospovirus Caper latent 1031708 Phietavirus X2 320850 Wigeon 1159908 virus coronavirus HKU20 Capim 1933265 Phifelvirus 1633149 Wild cucumber 70824 orthobunyavirus FL1 mosaic virus Capistrivirus 2011077 Phikmvvirus 2733349 Wild melon KSF1 15pyo banding virus Capraria 2049955 Phlox virus S 436066 Wild onion 1862127 yellow spot symptomless virus virus Caprine 39944 Phnom Penh 64894 Wild potato 187977 alphaherpesvirus bat virus mosaic virus 1 Caprine 11660 Phocid 47418 Wild tomato 400396 arthritis alphaherpesvirus mosaic virus encephalitis 1 virus Caprine 135102 Phocid 47419 Wild Vitis latent 2560839 gammaherpes gammaherpes virus virus 2 virus 2 Caprine 2560372 Phocid 2560643 Wilnyevirus 2560486 respirovirus 3 gammaherpes billnye virus 3 Capsicum 2560373 Phocine 11240 Wilsonroadvirus 2734007 chlorosis morbillivirus Sd1 orthotospovirus Capsicum 2734586 Pholetesor Winged bean 2169693 India ornigis alphaendornavirus alphasatellite bracovirus 1 Captovirus 235266 Phthorimaea 192584 Winklervirus 2560752 AFV1 operculella chi14 granulovirus Capuchin 2163996 Phutvirus 2733655 Wiseana signata 65124 monkey PPpW4 nucleopolyhedro hepatitis B virus virus Caraparu 1933290 Phyllosphere Wissadula golden 51673 orthobunyavirus sclerotimonavirus mosaic virus Carbovirus 2136037 Physalis 72539 Wissadula yellow 1904884 queenslandense mottle virus mosaic virus Dyonupapillo 1513250 Physarum Wisteria 1973265 mavirus 1 polycephalum badnavirus 1 Tp1 virus Dyoomegapap 1918731 Phytophthora 310750 Wisteria vein 201862 illomavirus 1 alphaendornavirus mosaic virus 1 Dyoomikronp 1513251 Picardvirus 2734264 Witwatersrand 2560841 apillomavirus picard orthobunyavirus 1 Dyophipapillo 1920493 Pidgey 2509390 Wizardvirus 2170253 mavirus 1 pidchovirus twister6 Dyopipapillo 1513252 Piedvirus 2733947 Wizardvirus 2170254 mavirus 1 IMEDE1 wizard Dyopsipapillo 1920498 Pienvirus 2733373 Woesvirus woes 1982751 mavirus 1 R801 Dyorhopapillo 1513253 Pifdecavirus 2733657 Wolkberg 2170059 mavirus 1 IBBPF7A orthobunyavirus Dyosigmapapi 1513254 Plum bark 675077 Wongorr virus 47465 llomavirus 1 necrosis stem pitting- associated virus Dyotaupapillo 1932910 Plum pox 12211 Wongtaivirus 2169922 mavirus 1 virus HK542 Dyothetapapill 1235662 Plumeria 1501716 Woodchuck 35269 omavirus 1 mosaic virus hepatitis virus Dyoupsilonpa 1932912 Plutella 98383 Woodruffvirus 1982746 pillomavirus 1 xylostella TP1604 granulovirus Dyoxipapillo 1513255 Poa semilatent 12328 Woodruffvirus 1982747 mavirus 1 virus YDN12 Dyoxipapillo 2169881 Poaceae 1985392 Woolly monkey 68416 mavirus 2 associated hepatitis B virus gemycircularvirus 1 Dyozetapapill 1177766 Podivirus 2733948 Woolly monkey 11970 omavirus 1 S05C243 sarcoma virus Eapunavirus 2733615 Poecivirus A 2560644 Wound tumor 10987 Eap1 virus East African 223262 Pogseptimavirus 2733996 Wphvirus 2560329 cassava PG07 BPS10C mosaic Cameroon virus East African 393599 Pogseptimavirus 2733997 Wphvirus BPS13 1987727 cassava VspSw1 mosaic Kenya virus East African 223264 Poindextervirus 2734196 Wphvirus hakuna 1987729 cassava BL10 mosaic Malawi virus East African 62079 Poindextervirus 2748760 Wphvirus 1987728 cassava rogue megatron mosaic virus East African 223275 Poinsettia 305785 Wphvirus WPh 1922328 cassava latent virus mosaic Zanzibar virus East Asian 2734556 Poinsettia 113553 Wuchang 1980542 Passiflora mosaic virus cockroach distortion orthophasmavirus virus 1 East Asian 341167 Pokeweed 1220025 Wuhan mivirus 2507319 Passiflora mosaic virus virus Eastern 2170195 Pokrovskaiavirus 2733374 Wuhan mosquito 1980543 chimpanzee fHe Yen301 orthophasmavirus simian foamy 1 virus Eastern equine 11021 Pokrovskaiavirus 2733375 Wuhan mosquito 1980544 encephalitis pv8018 orthophasmavirus virus 2 Eastern 2734571 Polar bear Wuhanvirus 2733969 kangaroopox mastadenovirus PHB01 virus A Eastlansingvirus 2734004 Pollockvirus 2170215 Wuhanvirus 2733970 Sf12 pollock PHB02 Echarate 2734447 Pollyceevirus 2560679 Wumivirus 2509286 phlebovirus pollyC millepedae Echinochloa 42630 Polybotosvirus 2560286 Wumpquatrovirus 400567 hoja blanca Atuph07 WMP4 tenuivirus Echinochloa Polygonum 430606 Wumptrevirus 440250 ragged stunt ringspot WMP3 virus orthotospovirus Eclipta yellow 2030126 Pomona bat 2049933 Wutai mosquito 1980612 vein hepatitis B phasivirus alphasatellite virus Eclipta yellow 875324 Pongine 159603 Wyeomyia 273350 vein virus gammaherpes orthobunyavirus virus 2 Eclunavirus 2560414 Poplar mosaic 12166 Xanthophyllomy 1167690 EcL1 virus ces dendrorhous virus L1A Ectocarpus 2083183 Popoffvirus 2560283 Xanthophyllomy 1167691 fasciculatus pv56 ces dendrorhous virus a virus L1B Ectocarpus 37665 Porcine 1985393 Xapuri 2734417 siliculosus associated mammarenavirus virus 1 gemycircularvirus 1 Ectocarpus Potato virus Y 12216 Xestia c-nigrum 51677 siliculosus granulovirus virus a Ectromelia 12643 Potato yellow 2230887 Xiamenvirus 1982373 virus blotch virus RDJL1 Ectropis 59376 Potato yellow 223307 Xiamenvirus 1982374 obliqua mosaic RDJL2 nucleopolyhed Panama virus rovirus Ectropis 1225732 Potato yellow 10827 Xilang striavirus 2560844 obliqua virus mosaic virus Edenvirus 2734230 Potato yellow 103881 Xinzhou mivirus 2507320 eden vein virus Edge Hill 64296 Pothos latent 44562 Xipapillomavirus 10561 virus virus 1 Efquatrovirus 2560415 Potosi 2560646 Xipapillomavirus 1513273 AL2 orthobunyavirus 2 Efquatrovirus 2560416 Poushouvirus 2560396 Yokohamavirus 1980942 AL3 Poushou PEi21 Efquatrovirus 2560417 Pouzolzia 1225069 Yokose virus 64294 AUEF3 golden mosaic virus Efquatrovirus 2560424 Primate T- 194443 Yoloswagvirus 2734158 EcZZ2 lymphotropic yoloswag virus 3 Efquatrovirus 2560420 Primolicivirus 2011081 Yongjia 2734607 EF3 Pf1 uukuvirus Efquatrovirus 2560421 Primula 1511840 Youcai mosaic 228578 EF4 malacoides virus virus 1 Efquatrovirus 2560425 Priunavirus 2560652 Yunnan orbivirus 306276 EfaCPT1 PR1 Efquatrovirus 2560426 Privet ringspot 2169960 Yushanvirus 2733978 IME196 virus Spp001 Efquatrovirus 2560427 Prochlorococcus Yushanvirus 2733979 LY0322 virus SppYZU05 PHM1 Efquatrovirus 2560428 Prospect Hill 1980485 Yuyuevirus 2508254 PMBT2 orthohantavirus beihaiense Efquatrovirus 2560429 Protapanteles Yuyuevirus 2508255 SANTOR1 paleacritae shaheense bracovirus Efquatrovirus 2560430 Providence 213633 Zaire ebolavirus 186538 SHEF2 virus Efquatrovirus 2560431 Prune dwarf 33760 Zaliv Terpeniya 2734608 SHEF4 virus uukuvirus Efquatrovirus 2560432 Prunus latent 2560653 Zantedeschia 270478 SHEF5 virus mild mosaic virus Eganvirus EtG 2734059 Prunus 37733 Zarhavirus 2734410 necrotic zahedan ringspot virus Eganvirus 29252 Przondovirus 2733672 Zika virus 64320 ev186 KN31

The cascade assays described herein are particularly well-suited for simultaneous testing of multiple targets. Pools of two to 10,000 target nucleic acids of interest may be employed, e.g., pools of 2-1,000, 2-100, 2-50, or 2-10 target nucleic acids of interest. Further testing may be used to identify the specific member of the pool, if warranted.

While the methods described herein do not require the target nucleic acid of interest to be DNA (and in fact it is specifically contemplated that the target nucleic acid of interest may be RNA), it is understood by those in the field that a reverse transcription step to convert target RNA to cDNA may be performed prior to or while contacting the biological sample with the composition.

Nucleic Acid-Guided Nucleases

The cascade assays comprise nucleic acid-guided nucleases in the reaction mix, either provided as a protein, a coding sequence for the protein, or, in many embodiments, in a ribonucleoprotein (RNP) complex. In some embodiments, the one or more nucleic acid-guided nucleases in the reaction mix may be, for example, a Cas nucleic acid-guided nuclease. Any nucleic acid-guided nuclease having both cis- and trans-cleavage activity may be employed, and the same nucleic acid-guided nuclease may be used for both RNP complexes or different nucleic acid-guided nucleases may be used in RNP1 and RNP2. For example, RNP1 and RNP2 may both comprise Cas12a nucleic acid-guided nucleases, or RNP1 may comprise a Cas13 nucleic acid-guided nuclease and RNP2 may comprise a Cas12a nucleic acid-guided nuclease or vice versa. In embodiments where a variant nucleic acid-guided nuclease is employed, only RNP2 will comprise the variant, and RNP1 may comprise either a Cas12a or Cas13 nucleic acid-guided nuclease. In embodiments where a variant nucleic acid-guided nuclease is not employed, either or both RNP1 and RNP2 can comprise a Cas13 nucleic acid-guided nuclease. Note that trans-cleavage activity is not triggered unless and until cis-cleavage activity (i.e., sequence specific activity) is initiated. Nucleic acid-guided nucleases include Type V and Type VI nucleic acid-guided nucleases, as well as nucleic acid-guided nucleases that comprise a RuvC nuclease domain or a RuvC-like nuclease domain but lack an HNH nuclease domain. Nucleic acid-guided nucleases with these properties are reviewed in Makarova and Koonin, Methods Mol. Biol., 1311:47-75 (2015) and Koonin, et al., Current Opinion in Microbiology, 37:67-78 (2020) and updated databases of nucleic acid-guided nucleases and nuclease systems that include newly-discovered systems include BioGRID ORCS (orcs:thebiogrid.org); GenomeCRISPR (genomecrispr.org); Plant Genome Editing Database (plantcrispr.org) and CRISPRCasFinder (crispercas.i2bc.paris-saclay.fr).

The type of nucleic acid-guided nuclease utilized in the method of detection depends on the type of target nucleic acid of interest to be detected. For example, a DNA nucleic acid-guided nuclease (e.g., a Cas12a, Cas14a, or Cas3) should be utilized if the target nucleic acid of interest is a DNA molecule, and an RNA nucleic acid-guided nuclease (e.g., Cas13a or Cas12g) should be utilized if the target nucleic acid of interest is an RNA molecule. Exemplary nucleic acid-guided nucleases include, but are not limited to, Cas RNA-guided DNA nucleic acid-guided nucleases, such as Cas3, Cas12a (e.g., AsCas12a, LbCas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, and Cas12j; Cas RNA-guided RNA nucleic acid-guided nucleases, such as Cas13a (LbaCas13, LbuCas13, LwaCas13), Cas13b (e.g., CccaCas13b, PsmCas13b), and Cas12g; and any other nucleic acid (DNA, RNA, or cDNA) targeting nucleic acid-guided nuclease with cis-cleavage activity and collateral trans-cleavage activity. In some embodiments, the nucleic acid-guided nuclease is a Type V CRISPR-Cas nuclease, such as Cas12a, Cas13a, or Cas14a. In some embodiments, the nucleic acid-guided nuclease is a Type I CRISPR-Cas nuclease, such as Cas3. Type II and Type VI nucleic acid-guided nucleases may also be employed.

In an RNP with a single crRNA (i.e., lacking/without a tracrRNA), Cas12a nucleases and related homologs and orthologs interact with a PAM (protospacer adjacent motif) sequence in a target nucleic acid for dsDNA unwinding and R-loop formation. Cas12a nucleases employ a multistep mechanism to ensure accurate recognition of spacer sequences in the target nucleic acid. The WED, REC1 and PAM-interacting (PI) domains of Cas12a nucleases are responsible for PAM recognition and for initiating invasion of the crRNA in the target dsDNA and for R-loop formation. It has been hypothesized that a conserved lysine residue is inserted into the dsDNA duplex, possibly initiating template strand/non-template strand unwinding. (See Jinek, et al, Mol. Cell, 73(3):589-600.e4 (2019).) PAM binding further introduces a kink in the target strand, which further contributes to local strand separation and facilitates base paring of the target strand to the seed segment of the crRNA while the displaced non-target strand is stabilized by interactions with the PAM-interacting domains. (Id.) The variant nucleic acid-guided nucleases disclosed herein and discussed in detail below have been engineered to disrupt one or both of the WED and PI domains to reconfigure the site of unwinding and R-loop formation to, e.g., sterically obstruct dsDNA target nucleic acids from binding to the variant nucleic acid-guided nuclease and/or to minimize strand separation and/or stabilization of the non-target strand. Though contrary to common wisdom, engineering the variant nucleic acid-guided nucleases in this way contributes to a robust and high-fidelity cascade assay.

The variant nucleic acid-guided nucleases disclosed herein are variants of wildtype Type V nucleases LbCas12a (Lachnospriaceae bacterium Cas12a), AsCas 12a (Acidaminococcus sp. BV3L6 Cas12a), CtCas12a (Candidatus Methanoplasma termitum Cas12a), EeCas12a (Eubacterium eligens Cas12a), Mb3Cas12a (Moraxella bovoculi Cas12a), FnCas12a (Francisella novicida Cas12a), FnoCas12a (Francisella tularensis subsp. novicida FTG Cas12a), FbCas12a (Flavobacteriales bacterium Cas12a), Lb4Cas12a (Lachnospira eligens Cas12a), MbCas12a (Moraxella bovoculi Cas12a), Pb2Cas12a (Prevotella bryantii Cas12a), PgCas12a (Candidatus Parcubacteria bacterium Cas12a), AaCas12a (Acidaminococcus sp. Cas12a), BoCas12a (Bacteroidetes bacterium Cas12a), CMaCas12a (Candidatus Methanomethylophilus alvus CMx1201 Cas12a), and to-be-discovered equivalent Cas12a nucleic acid-guided nucleases and homologs and orthologs of these nucleic acid-guided nucleases (and other nucleic acid-guided nucleases that exhibit both cis-cleavage and trans-cleavage activity), where mutations have been made to the PAM interacting domains such that double-stranded DNA (dsDNA) substrates are bound much more slowly to the variant nucleic acid-guided nucleases than to their wildtype nucleic acid-guided nuclease counterpart, yet single-stranded DNA (ssDNA) substrates are bound at the same rate or nearly so as their wildtype nucleic acid-guided nuclease counterpart. The variant nucleic acid-guided nucleases comprise reconfigured domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules to achieve this phenotype and are described in detail below.

Guide RNA (gRNA)

The present disclosure detects a target nucleic acid of interest via a reaction mixture containing at least two guide RNAs (gRNAs) each incorporated into a different RNP complex (i.e., RNP1 and RNP2). Suitable gRNAs include at least one crRNA region to enable specificity in every reaction. The gRNA of RNP1 is specific to a target nucleic acid of interest and the gRNA of RNP2 is specific to an unblocked nucleic acid or a synthesized activating molecule (both described in detail below). As will be clear given the description below, an advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (i.e., the gRNA specific to the target nucleic acid of interest), the cascade assay components can stay the same (i.e., are identical or substantially identical) no matter what target nucleic acid(s) of interest are being detected, and the gRNA in RNP1 is easily reprogrammable.

Like the nucleic acid-guided nuclease, the gRNA may be provided in the cascade assay reaction mix in a preassembled RNP, as an RNA molecule, or may also be provided as a DNA sequence to be transcribed, in, e.g., a vector backbone. Providing the gRNA in a pre-assembled RNP complex (i.e., RNP1 or RNP2) is preferred if rapid kinetics are preferred. If provided as a gRNA molecule, the gRNA sequence may include multiple endoribonuclease recognition sites (e.g., Csy4) for multiplex processing. Alternatively, if provided as a DNA sequence to be transcribed, an endoribonuclease recognition site may be encoded between neighboring gRNA sequences such that more than one gRNA can be transcribed in a single expression cassette. Direct repeats can also serve as endoribonuclease recognition sites for multiplex processing. Guide RNAs are generally about 20 nucleotides to about 300 nucleotides in length and may contain a spacer sequence containing a plurality of bases and complementary to a protospacer sequence in the target sequence. The gRNA spacer sequence may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more complementary to its intended target nucleic acid of interest.

The gRNA of RNP1 is capable of complexing with the nucleic acid-guided nuclease of RNP1 to perform cis-cleavage of a target nucleic acid of interest (e.g., a DNA or RNA), which triggers non-sequence specific trans-cleavage of other molecules in the reaction mix. Guide RNAs include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest (or target sequences generated by unblocking blocked nucleic acid molecules or target sequences generated by synthesizing synthesized activating molecules as described below). Target nucleic acids of interest (describe in detail above) preferably include a protospacer-adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region of the target nucleic acid of interest.

In some embodiments, the gRNA (e.g., of RNP1) is an exo-resistant circular molecule that can include several DNA bases between the 5′ end and the 3′ end of a natural guide RNA and is capable of binding a target sequence. The length of the circularized guide for RNP1 can be such that the circular form of guide can be complexed with a nucleic acid-guided nuclease to form a modified RNP1 which can still retain its cis-cleavage i.e., (specific) and trans-cleavage (i.e., non-specific) nuclease activity.

In any of the foregoing embodiments, the gRNA may be a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the gRNAs of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). By way of further example, a modified nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and/or internucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described herein.

Ribonucleoprotein (RNP) Complex

As described above, although the cascade assay “reaction mix” may comprise separate nucleic acid-guided nucleases and gRNAs (or coding sequences therefor), the cascade assays preferably comprise preassembled ribonucleoprotein complexes (RNPs) in the reaction mix, allowing for faster detection kinetics. The present cascade assay employs at least two types of RNP complexes—RNP1 and RNP2—each type containing a nucleic acid-guided nuclease and a gRNA. RNP1 and RNP2 may comprise the same nucleic acid-guided nuclease or may comprise different nucleic acid-guided nucleases; however, the gRNAs in RNP1 and RNP2 are different and are configured to detect different nucleic acids. In some embodiments, the reaction mixture contains about 1 fM to about 10 μM of a given RNP1, or about 1 μM to about 1 μM of a given RNP1, or about 10 μM to about 500 μM of a given RNP1. In some embodiments the reaction mixture contains about 6×10⁴ to about 6×10¹² complexes per microliter (μl) of a given RNP1, or about 6×10⁶ to about 6×10¹⁰ complexes per microliter (μl) of a given RNP1. In some embodiments, the reaction mixture contains about 1 fM to about 500 μM of a given RNP2, or about 1 μM to about 250 μM of a given RNP2, or about 10 μM to about 100 μM of a given RNP2. In some embodiments the reaction mixture contains about 6×10⁴ to about 6×10¹² complexes per microliter (μl) of a given RNP2 or about 6×10⁶ to about 6×10¹² complexes per microliter (μl) of a given RNP2. See Example II below describing preassembling RNPs and Examples V and VI below describing various cascade assay conditions where the relative concentrations of RNP2 and the blocked nucleic acid molecules is adjusted as described below.

In any of the embodiments of the disclosure, the reaction mixture includes 1 to about 1,000 different RNP1s (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 10,000 or more RNP1s), where different RNP1s comprise a different gRNA (or crRNA thereof) polynucleotide sequence. For example, a reaction mixture designed for environmental or oncology testing comprises more than one unique RNP1-gRNA (or RNP1-crRNA) ribonucleoprotein complex for the purpose of detecting more than one target nucleic acid of interest. That is, more than one RNP1 may also be present for the purpose of targeting one target nucleic acid of interest from many sources or for targeting more than one target nucleic acid of interest from a single source.

In any of the foregoing embodiments, the gRNA of RNP1 may be homologous or heterologous, relative to the gRNA of other RNP1(s) present in the reaction mixture. A homologous mixture of RNP1 gRNAs has a number of gRNAs with the same nucleotide sequence, whereas a heterologous mixture of RNP1 gRNAs has multiple gRNAs with different nucleotide sequences (e.g., gRNAs targeting different loci, genes, variants, and/or microbial species). Therefore, the disclosed methods of identifying one or more target nucleic acids of interest may include a reaction mixture containing more than two heterologous gRNAs, more than three heterologous gRNAs, more than four heterologous gRNAs, more than five heterologous gRNAs, more than six heterologous gRNAs, more than seven heterologous gRNAs, more than eight heterologous gRNAs, more than nine heterologous gRNAs, more than ten heterologous gRNAs, more than eleven heterologous gRNAs, more than twelve heterologous gRNAs, more than thirteen heterologous gRNAs, more than fourteen heterologous gRNAs, more than fifteen heterologous gRNAs, more than sixteen heterologous gRNAs, more than seventeen heterologous gRNAs, more than eighteen heterologous gRNAs, more than nineteen heterologous gRNAs, more than twenty heterologous gRNAs, more than twenty-one heterologous gRNAs, more than twenty-three heterologous gRNAs, more than twenty-four heterologous gRNAs, or more than twenty-five heterologous gRNAs. Such a heterologous mixture of RNP1 gRNAs in a single reaction enables multiplex testing.

As a first non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNP1s (RNP1-1s) having a gRNA targeting parainfluenza virus 1; a number of RNP1s (RNP1-2s) having a gRNA targeting human metapneumovirus; a number of RNP1s (RNP1-3s) having a gRNA targeting human rhinovirus; a number of RNP1s (RNP1-4s) having a gRNA targeting human enterovirus; and a number of RNP1s (RNP1-5s) having a gRNA targeting coronavirus HKU1. As a second non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNP1s containing a gRNA targeting two or more SARS-Co-V-2 variants, e.g., B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, and BA.5 and subvariants thereof.

As another non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain RNP1s targeting two or more target nucleic acids of interest from organisms that infect grapevines, such as Guignardia bidwellii (RNP1-1), Uncinula necator (RNP1-2), Botrytis cincerea (RNP1-3), Plasmopara viticola (RNP1-4), and Botryotinis fuckleina (RNP1-5).

Reporter Moieties

The cascade assay detects a target nucleic acid of interest via detection of a signal generated in the reaction mix by a reporter moiety. In some embodiments the detection of the target nucleic acid of interest occurs virtually instantaneously. For example, see the results reported in Example VI for assays comprising 3e4 or 30 copies of MRSA target and within 1 minute or less at 3 copies of MRSA target (see, e.g., FIGS. 10B-10H). Reporter moieties can comprise DNA, RNA, a chimera of DNA and RNA, and can be single stranded, double stranded, or a moiety that is a combination of single stranded portions and double stranded portions.

Depending on the type of reporter moiety used, trans- and/or cis-cleavage by the nucleic acid-guided nuclease in RNP2 releases a signal. In some embodiments, trans-cleavage of stand-alone reporter moieties (e.g., not bound to any blocked nucleic acid molecules or blocked primer molecules) may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time (shown in FIG. 1B and at top of FIG. 4 ). Trans-cleavage by either an activated RNP1 or an activated RNP2 may release a signal. In alternative embodiments and preferably, the reporter moiety may be bound to the blocked nucleic acid molecule, where trans-cleavage of the blocked nucleic acid molecule (or blocked primer molecule) and conversion to an unblocked nucleic acid molecule (or unblocked primer molecule) may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time, thus allowing for real time reporting of results (shown at FIG. 4 , center). In yet another embodiment, the reporter moiety may be bound to a blocked nucleic acid molecule such that cis-cleavage following the binding of the RNP2 to an unblocked nucleic acid molecule releases a PAM distal sequence, which in turn generates a signal at rates that are proportional to the cleavage rate (shown at FIG. 4 , bottom). In this case, activation of RNP2 by cis- (target specific) cleavage of the unblocked nucleic acid molecule directly produces a signal, rather than producing a signal via indiscriminate trans-cleavage activity. Alternatively or in addition, a reporter moiety may be bound to the gRNA.

The reporter moiety may be a synthetic molecule linked or conjugated to a reporter and quencher such as, for example, a TaqMan probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The reporter and quencher may be about 20-30 bases apart or less (i.e., 10-11 nm apart or less) for effective quenching via fluorescence resonance energy transfer (FRET). Alternatively, signal generation may occur through different mechanisms. Other detectable moieties, labels, or reporters can also be used to detect a target nucleic acid of interest as described herein. Reporter moieties can be labeled in a variety of ways, including direct or indirect attachment of a detectable moiety such as a fluorescent moiety, hapten, or colorimetric moiety.

Examples of detectable moieties include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, and protein-protein binding pairs, e.g., protein-antibody binding pairs. Examples of fluorescent moieties include, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansyl chloride, phycocyanin, and phycoerythrin. Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, and aequorin. Examples of enzyme systems having visually detectable signals include, but are not limited to, galactosidases, glucuronidases, phosphatases, peroxidases, and cholinesterases. Identifiable markers also include radioactive elements such as ¹²⁵1, ³⁵S, ¹⁴C, or ³H. Reporters can also include a change in pH or charge of the cascade assay reaction mix.

The methods used to detect the generated signal will depend on the reporter moiety or moieties used. For example, a radioactive label can be detected using a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging. Fluorescent labels can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Simple colorimetric labels can be detected by observing the color associated with the label. When pairs of fluorophores are used in an assay, fluorophores are chosen that have distinct emission patterns (wavelengths) so that they can be easily distinguished. In some embodiments, the signal can be detected by lateral flow assays (LFAs). Lateral flow tests are simple devices intended to detect the presence or absence of a target nucleic acid of interest in a sample. LFAs can use nucleic acid molecules conjugated nanoparticles (often gold, e.g., RNA-AuNPs or DNA-AuNPs) as a detection probe, which hybridizes to a complementary target sequence. (See FIG. 9 and the description thereof below.) The classic example of an LFA is the home pregnancy test.

Single-stranded, double-stranded or reporter moieties comprising both single- and double-stranded portions can be introduced to show a signal change proportional to the cleavage rate, which increases with every new activated RNP2 complex over time. In some embodiments and as described in detail below, reporter moieties can also be embedded into the blocked nucleic acid molecules (or blocked primer molecules) for real time reporting of results.

For example, the method of detecting a target nucleic acid molecule in a sample using a cascade assay as described herein can involve contacting the reaction mix with a labeled detection ssDNA containing a fluorescent resonance energy transfer (FRET) pair, a quencher/phosphor pair, or both. A FRET pair consists of a donor chromophore and an acceptor chromophore, where the acceptor chromophore may be a quencher molecule. FRET pairs (donor/acceptor) suitable for use include, but are not limited to, EDANS/fluorescein, IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL, fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5, Texas Red/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL, and fluorescein/LC Red 705. In addition, a fluorophore/quantum dot donor/acceptor pair can be used. EDANS is (5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid); IAEDANS is 5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid); DABCYL is 4-(4-dimethylaminophenyl)diazenylbenzoic acid. Useful quenchers include, but are not limited to, BHQ, DABCYL, QSY 7 and QSY 33.

In any of the foregoing embodiments, the reporter moiety may comprise one or more modified nucleic acid molecules, containing a modified nucleoside or nucleotide. In some embodiments the modified nucleoside or nucleotide is chosen from 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described below.

Nucleic Acid Modifications

For any of the nucleic acid molecules described herein (e.g., blocked nucleic acid molecules, blocked primer molecules, gRNAs, template molecules, synthesized activating molecules, and reporter moieties), the nucleic acid molecules may be used in a wholly or partially modified form. Typically, modifications to the blocked nucleic acid molecules, gRNAs, template molecules, reporter moieties, and blocked primer molecules described herein are introduced to optimize the molecule's biophysical properties (e.g., increasing nucleic acid-guided nuclease resistance and/or increasing thermal stability). Modifications typically are achieved by the incorporation of, for example, one or more alternative nucleosides, alternative sugar moieties, and/or alternative internucleoside linkages.

For example, one or more of the cascade assay components may include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and/or 3-deazaguanine and 3-deazaadenine. The nucleic acid molecules described herein (e.g., blocked nucleic acid molecules, blocked primer molecules, gRNAs, reporter molecules, synthesized activating molecules, and template molecules) may also include nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and/or 2-pyridone. Further modification of the nucleic acid molecules described herein may include nucleobases disclosed in U.S. Pat. No. 3,687,808; Kroschwitz, ed., The Concise Encyclopedia of Polymer Science and Engineering, NY, John Wiley & Sons, 1990, pp. 858-859; Englisch, et al., Angewandte Chemie, 30:613 (1991); and Sanghvi, Chapter 16, Antisense Research and Applications, CRC Press, Gait, ed., 1993, pp. 289-302.

In addition to or as an alternative to nucleoside modifications, the cascade assay components may comprise 2′ sugar modifications, including 2′-O-methyl (2′-O-Me), 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE), 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and/or 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylamino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂OCH₂N(CH₃)₂. Other possible 2′-modifications that can modify the nucleic acid molecules described herein (i.e., blocked nucleic acid molecules, gRNAs, synthesized activating molecules, reporter molecules, and blocked primer molecules) may include all possible orientations of OH; F; O-, S-, or N-alkyl (mono- or di-); O-, S-, or N-alkenyl (mono- or di-); O-, S- or N-alkynyl (mono- or di-); or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Other potential sugar substituent groups include, e.g., aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) and fluoro (F). 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the interfering RNA molecule, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

Finally, modifications to the cascade assay components may comprise internucleoside modifications such as phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.

The Signal Boosting Cascade Assay Employing Blocked Nucleic Acid Molecules

Before getting to the details relating to addressing undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules), understanding the cascade assay itself is key. FIG. 1B, described above, depicts the cascade assay generally. A specific embodiment of the cascade assay utilizing blocked nucleic acid molecules is depicted in FIG. 2A and described in detail below. In this embodiment, a blocked nucleic acid is used to prevent the activation of RNP2 in the absence of a target nucleic acid of interest. The method in FIG. 2A begins with providing the cascade assay components RNP1 (201), RNP2 (202) and blocked nucleic acid molecules (203). RNP1 (201) comprises a gRNA specific for a target nucleic acid of interest and a nucleic acid-guided nuclease (e.g., Cas 12a or Cas 14 for a DNA target nucleic acid of interest or a Cas 13a for an RNA target nucleic acid of interest) and RNP2 (202) comprises a gRNA specific for an unblocked nucleic acid molecule and a nucleic acid-guided nuclease (again, e.g., Cas 12a or Cas 14 for a DNA unblocked nucleic acid molecule or a Cas 13a for an RNA unblocked nucleic acid molecule). As described above, the nucleic acid-guided nucleases in RNP1 (201) and RNP2 (202) can be the same or different depending on the type of target nucleic acid of interest and unblocked nucleic acid molecule. What is key, however, is that the nucleic acid-guided nucleases in RNP1 and RNP2 may be activated to have trans-cleavage activity following initiation of cis-cleavage activity.

In a first step, a sample comprising a target nucleic acid of interest (204) is added to the cascade assay reaction mix. The target nucleic acid of interest (204) combines with and activates RNP1 (205) but does not interact with or activate RNP2 (202). Once activated, RNP1 binds the target nucleic acid of interest (204) and cuts the target nucleic acid of interest (204) via sequence-specific cis-cleavage, activating non-specific trans-cleavage of other nucleic acids present in the reaction mix, including the blocked nucleic acid molecules (203). At least one of the blocked nucleic acid molecules (203) becomes an unblocked nucleic acid molecule (206) when the blocking moiety (207) is removed. As described below, “blocking moiety” may refer to nucleoside modifications, topographical configurations such as secondary structures, and/or structural modifications.

Once at least one of the blocked nucleic acid molecules (203) is unblocked, the unblocked nucleic acid molecule (206) can then bind to and activate an RNP2 (208). Because the nucleic acid-guided nucleases in the RNP1s (205) and RNP2s (208) have both cis- and trans-cleavage activity, the trans-cleavage activity causes more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering activation of even more RNP2s (208) and more trans-cleavage activity in a cascade. FIG. 2A at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (209) comprise a quencher (210) and a fluorophore (211) linked by a nucleic acid sequence. As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (205) and RNP2 (208). The intact reporter moieties (209) become activated reporter moieties (212) when the quencher (210) is separated from the fluorophore (211), emitting a fluorescent signal (213). Signal strength increases rapidly as more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering cis-cleavage activity of more RNP2s (208) and thus more trans-cleavage activity of the reporter moieties (209). Again, the reporter moieties are shown here as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4 . One particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (gRNA1), the cascade assay components are modular in the sense that the components stay the same no matter what target nucleic acid(s) of interest are being detected.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule (220) and an exemplary technique for unblocking the blocked nucleic acid molecules described herein. A blocked single-stranded or double-stranded, circular or linear, DNA or RNA molecule (220) comprising a target strand (222) may contain a partial hybridization with a complementary non-target strand nucleic acid molecule (224) containing unhybridized and cleavable secondary loop structures (226) (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Trans-cleavage of the loops by, e.g., activated RNP1s or RNP2s, generates short strand nucleotide sequences or regions (228) which, because of the short length and low melting temperature T_(m) can dehybridize at room temperature (e.g., 15°-25° C.), thereby unblocking the blocked nucleic acid molecule (220) to create an unblocked nucleic acid molecule (230), enabling the internalization of the unblocked nucleic acid molecule (230) (target strand) into an RNP2, leading to RNP2 activation.

A blocked nucleic acid molecule may be single-stranded or double-stranded, circular or linear, and may further contain a partially hybridized nucleic acid sequence containing cleavable secondary loop structures, as exemplified by “L” in FIGS. 2C-2E. Such blocked nucleic acid molecules typically have a low binding affinity, or high dissociation constant (K_(d)) in relation to binding to RNP2 and may be referred to herein as a high K_(d) nucleic acid molecule. In the context of the present disclosure, the binding of blocked or unblocked nucleic acid molecules or blocked or unblocked primer molecules to RNP2, low K_(d) values range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high K_(d) values are in the range of 100 nM to about 10-100 10 mM and thus are about 10⁵-10⁶-, 10⁷-, 10⁸-, 10⁹- to 10¹⁰-fold or higher as compared to low K_(d) values. Of course, the ideal blocked nucleic acid molecule would have an “infinite K_(d).”

The blocked nucleic acid molecules (high K_(d) molecules) described herein can be converted into unblocked nucleic acid molecules (low K_(d) molecules—also in relation to binding to RNP2) via cleavage of nuclease-cleavable regions (e.g., via active RNP1s and RNP2s). The unblocked nucleic acid molecule has a higher binding affinity for the gRNA in RNP2 than does the blocked nucleic acid molecule, although, as described below, there is some “leakiness” where some blocked nucleic acid molecules are able to interact with the gRNA in the RNP2 triggering undesired unwinding.

Once the unblocked nucleic acid molecule is bound to RNP2, the RNP2 activation triggers trans-cleavage activity, which in turn leads to more RNP2 activation by further cleaving blocked nucleic acid molecules, resulting in a positive feedback loop or cascade.

In embodiments where blocked nucleic acid molecules are linear and/or form a secondary structure, the blocked nucleic acid molecules may be single-stranded (ss) or double-stranded (ds) and contain a first nucleotide sequence and a second nucleotide sequence. The first nucleotide sequence has sufficient complementarity to hybridize to a gRNA of RNP2, and the second nucleotide sequence does not. The first and second nucleotide sequences of a blocked nucleic acid molecule may be on the same nucleic acid molecule (e.g., for single-strand embodiments) or on separate nucleic acid molecules (e.g., for double-strand embodiments). Trans-cleavage (e.g., via RNP1 or RNP2) of the second nucleotide sequence converts the blocked nucleic acid molecule to a single-strand unblocked nucleic acid molecule. The unblocked nucleic acid molecule contains only the first nucleotide sequence, which has sufficient complementarity to hybridize to the gRNA of RNP2, thereby activating the trans-cleavage activity of RNP2.

In some embodiments, the second nucleotide sequence at least partially hybridizes to the first nucleotide sequence, resulting in a secondary structure containing at least one loop (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Such loops block the nucleic acid molecule from binding or incorporating into an RNP complex thereby initiating cis- or trans-cleavage (see, e.g., the exemplary structures in FIGS. 2C-2F).

In some embodiments, the blocked nucleic acid molecule may contain a protospacer adjacent motif (PAM) sequence, or partial PAM sequence, positioned between the first and second nucleotide sequences, where the first sequence is 5′ to the PAM sequence, or partial PAM sequence, (see FIG. 2G). Inclusion of a PAM sequence may increase the reaction kinetics internalizing the unblocked nucleic acid molecule into RNP2 and thus decrease the time to detection. In other embodiments, the blocked nucleic acid molecule does not contain a PAM sequence.

In some embodiments, the blocked nucleic acid molecules (i.e., high K_(d) nucleic acid molecules in relation to binding to RNP2) of the disclosure may include a structure represented by Formula I (e.g., FIG. 2C), Formula II (e.g., FIG. 2D), Formula III (e.g., FIG. 2E), or Formula IV (e.g., FIG. 2F) wherein Formulas I-IV are in the 5′-to-3′ direction: A-(B-L)J-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;     -   B is 4-12 nucleotides in length;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10;     -   C is 4-15 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then A-(B-L)J-C and T-D are separate nucleic acid         strands;     -   T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or         17-25) and comprises a sequence complementary to B and C; and     -   D is 0-10 nucleotides in length and comprises a sequence         complementary to A;         D-T-T′-C-(L-B)J-A  (Formula II);     -   wherein D is 0-10 nucleotides in length;     -   T-T′ is 17-135 nucleotides in length (e.g., 17-100, 17-50, or         17-25);     -   T′ is 1-10 nucleotides in length and does not hybridize with T;     -   C is 4-15 nucleotides in length and comprises a sequence         complementary to T;     -   L is 3-25 nucleotides in length and does not hybridize with T;     -   B is 4-12 nucleotides in length and comprises a sequence         complementary to T;     -   J is an integer between 1 and 10;     -   A is 0-15 nucleotides in length and comprises a sequence         complementary to D;         T-D-M-A-(B-L)J-C(Formula III);     -   wherein T is 17-135 nucleotides in length (e.g., 17-100, 17-50,         or 17-25);     -   D is 0-10 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then T-D and A-(B-L)J-C are separate nucleic acid         strands;     -   A is 0-15 nucleotides in length and comprises a sequence         complementary to D;     -   B is 4-12 nucleotides in length and comprises a sequence         complementary to T;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10; and     -   C is 4-15 nucleotides in length;         T-D-M-A-L_(p)-C(Formula IV);     -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,         or 17-25);     -   D is 0-15 nucleotides in length;     -   M is 1-25 nucleotides in length;     -   A is 0-15 nucleotides in length and comprises a sequence         complementary to D; and     -   L is 3-25 nucleotides in length;     -   p is 0 or 1;     -   C is 4-15 nucleotides in length and comprises a sequence         complementary to T.         In alternative embodiments of any of these molecules, T (or         T-T′) can have a maximum length of 1,000 nucleotides, e.g., at         most 750, at most 500, at most 400, at more 300, at most 250, at         most 200, at most 150, at most 135, at most 100, at most 75, at         most 50, or at most 25 nucleotides.

Nucleotide mismatches can be introduced in any of the above structures containing double-strand segments (for example, where M is absent in Formula I or Formula III) to reduce the melting temperature (T_(m)) of the segment such that once the loop (L) is cleaved, the double-strand segment is unstable and dehybridizes rapidly. The percentage of nucleotide mismatches of a given segment may vary between 0% and 50%; however, the maximum number of nucleotide mismatches is limited to a number where the secondary loop structure still forms. “Segments” in the above statement refers to A, B, and C. In other words, the number of hybridized bases can be less than or equal to the length of each double-strand segment and vary based on number of mismatches introduced.

In any blocked nucleic acid molecule having the structure of Formula I, III, or IV, T will have sequence complementarity to a nucleotide sequence (e.g., a spacer sequence) within a gRNA of RNP2. The nucleotide sequence of T is to be designed such that hybridization of T to the gRNA of RNP2 activates the trans-nuclease activity of RNP2. In any blocked nucleic acid molecule having structure of Formula II, T-T′ will have sequence complementarity to a sequence (e.g., a spacer sequence) within the gRNA of RNP2. The nucleotide sequence of T-T′ is to be designed such that hybridization of T-T′ to the gRNA of RNP2 activates the trans-nuclease activity of RNP2. For T or T-T′, full complementarity to the gRNA is not necessarily required, provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of RNP2.

In any of the foregoing embodiments, the blocked nucleic acid molecules of the disclosure may and preferably do further contain a reporter moiety attached thereto such that cleavage of the blocked nucleic acid releases a signal from the reporter moiety. (See FIG. 4 , mechanisms depicted at center and bottom.)

Also, in any of the foregoing embodiments, the blocked nucleic acid molecule may be a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the blocked nucleic acid molecules of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). The blocked nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and/or internucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, any other nucleic acid molecule modifications described above, and any combination thereof.

FIG. 2G at left shows an exemplary single-strand blocked nucleic acid molecule and how the configuration of this blocked nucleic acid molecule is able to prevent (or significantly prevent) undesired unwinding of the blocked nucleic acid molecule (or blocked primer molecule) and R-loop formation with an RNP complex, thereby blocking activation of the trans-cleavage activity of RNP2. The single-strand blocked nucleic acid molecule is self-hybridized and comprises: a target strand (TS) sequence complementary to the gRNA (e.g., crRNA) of RNP2; a cleavable non-target strand (NTS) sequence that is partially hybridized (e.g., it contains secondary loop structures) to the TS sequence; and a protospacer adjacent motif (PAM) sequence (e.g., 5′ NAAA 3′) that is specifically located at the 3′ end of the TS sequence. An RNP complex with 3′→5′ diffusion (e.g., 1D diffusion) initiates R-loop formation upon PAM recognition. R-loop formation is completed upon a stabilizing ≥17 base hybridization of the TS to the gRNA of RNP2; however, because of the orientation of the PAM sequence relative to the secondary loop structure(s), the blocked nucleic acid molecule sterically prevents the target strand from hybridizing with the gRNA of RNP2, thereby blocking the stable R-loop formation required for the cascade reaction.

FIG. 2G at right shows the blocked nucleic acid molecule being unblocked via trans-cleavage (e.g., by RNP1) and subsequent dehybridization of the non-target strand's secondary loop structures, followed by binding of the target strand to the gRNA of RNP2, thereby completing stable R-loop formation and activating the trans-cleavage activity of the RNP2 complex.

In some embodiments, the blocked nucleic acid molecules provided herein are circular DNAs, RNAs or chimeric (DNA-RNA) molecules (FIG. 2H), and the blocked nucleic acid molecules may include different base compositions depending on the Cas enzyme used for RNP1 and RNP2. For the circular design of blocked nucleic acid molecules, the 5′ and 3′ ends are covalently linked together. This configuration makes internalization of the blocked nucleic acid molecule into RNP2—and subsequent RNP2 activation—sterically unfavorable, thereby blocking the progression of the cascade assay. Thus, RNP2 activation (e.g., trans-cleavage activity) happens after cleavage of a portion of the blocked nucleic acid molecule followed by linearization and internalization of unblocked nucleic acid molecule into RNP2.

In some embodiments, the blocked nucleic acid molecules are topologically circular molecules with 5′ and 3′ portions hybridized to each other using DNA, RNA, LNA, BNA, or PNA bases which have a very high melting temperature (Tm). The high Tm causes the structure to effectively behave as a circular molecule even though the 5′ and 3′ ends are not covalently linked. The 5′ and 3′ ends can also have base non-naturally occurring modifications such as phosphorothioate bonds to provide increased stability.

In embodiments where the blocked nucleic acid molecules are circularized (e.g., circular or topologically circular), as illustrated in FIG. 2H, each blocked nucleic acid molecule includes a first region, which is a target sequence specific to the gRNA of RNP2, and a second region, which is a sequence that can be cleaved by nuclease enzymes of activated RNP1 and/or RNP2. The first region may include a nuclease-resistant nucleic acid sequence such as, for example, a phosphorothioate group or other non-naturally occurring nuclease-resistant base modifications, for protection from trans-nucleic acid-guided nuclease activity. In some embodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas12a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant DNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence. In other embodiments, when the Cas enzyme in RNP1 is Cas12a and the Cas enzyme in RNP2 is Cas13a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant RNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence and a cleavable RNA sequence. In yet other embodiments, when the Cas enzyme in RNP1 is Cas13a and the Cas enzyme in RNP2 is Cas12a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant DNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence and a cleavable RNA sequence. In some other embodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas13a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant RNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable RNA sequence.

The Signal Boosting Cascade Assay Employing Blocked Primer Molecules

The blocked nucleic acid molecules described above may also be blocked primer molecules. Blocked primer molecules include a sequence complementary to a primer binding domain (PBD) on a template molecule (see description below in reference to FIGS. 3A and 3B) and can have the same general structures as the blocked nucleic acid molecules described above. A PBD serves as a nucleotide sequence for primer hybridization followed by primer polymerization by a polymerase. In any of Formulas I, II, or III described above, the blocked primer nucleic acid molecule may include a sequence complementary to the PBD on the 5′ end of T. The unblocked primer nucleic acid molecule can bind to a template molecule at the PBD and copy the template molecule via polymerization by a polymerase.

Specific embodiments of the cascade assay which utilize blocked primer molecules and are depicted in FIGS. 3A and 3B. In the embodiments using blocked nucleic acid molecules described above, activation of RNP1 by binding of N nucleotides of the target nucleic acid molecules or cis-cleavage of the target nucleic acid molecules initiates trans-cleavage of the blocked nucleic acid molecules which were used to activate RNP2—that is, the unblocked nucleic acid molecules are a target sequence for the gRNA in RNP2. In contrast, in the embodiments using blocked primers activation of RNP1 and trans-cleavage unblocks a blocked primer molecule that is then used to prime a template molecule for extension by a polymerase, thereby synthesizing synthesized activating molecules that are the target sequence for the gRNA in RNP2.

FIG. 3A is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and linear template molecules. At left of FIG. 3A is a cascade assay reaction mix comprising 1) RNP1s (301) (only one RNP1 is shown); 2) RNP2s (302); 3) linear template molecules (330) (which is the non-target strand); 4) a circular blocked primer molecule (334) (i.e., a high K_(d) molecule); and 5) a polymerase (338), such as a (D29 polymerase. The linear template molecule (330) (non-target strand) comprises a PAM sequence (331), a primer binding domain (PBD) (332) and, optionally, a nucleoside modification (333) to protect the linear template molecule (330) from 3′→5′ exonuclease activity. Blocked primer molecule (334) comprises a cleavable region (335) and a complement to the PBD (332) on the linear template molecule (330).

Upon addition of a sample comprising a target nucleic acid of interest (304) (capable of complexing with the gRNA in RNP1 (301)), the target nucleic acid of interest (304) is bound by with and activates RNP1 (305) but does not interact with or activate RNP2 (302). Once activated, RNP1 cuts the target nucleic acid of interest (304) via sequence specific cis-cleavage, which activates non-specific trans-cleavage of other nucleic acids present in the reaction mix, including at least one of the blocked primer molecules (334). The circular blocked primer molecule (334) (i.e., a high K_(d) molecule, where high K_(d) relates to binding to RNP2) upon cleavage becomes an unblocked linear primer molecule (344) (a low K_(d) molecule, where low K_(d) relates to binding to RNP2), which has a region (336) complementary to the PBD (332) on the linear template molecule (330) and can bind to the linear template molecule (330).

Once the unblocked linear primer molecule (344) and the linear template molecule (330) are hybridized (i.e., hybridized at the PBD (332) of the linear template molecule (330) and the PBD complement (336) on the unblocked linear primer molecule (344)), 3′→5′ exonuclease activity of the polymerase (338) removes the unhybridized single-stranded DNA at the end of the unblocked primer molecule (344) and the polymerase (338) can copy the linear template molecule (330) to produce a synthesized activating molecule (346) which is a complement of the non-target strand, which is the target strand. The synthesized activating molecule (346) is capable of activating RNP2 (302→308). As described above, because the nucleic acid-guided nuclease in the RNP2 (308) complex exhibits (that is, possesses) both cis- and trans-cleavage activity, more blocked primer molecules (334) become unblocked primer molecules (344) triggering activation of more RNP2s (308) and more trans-cleavage activity in a cascade. As stated above in relation to blocked and unblocked nucleic acid molecules (both linear and circular), the unblocked primer molecule has a higher binding affinity for the gRNA in RNP2 than does the blocked primer molecule, although there may be some “leakiness” where some blocked primer molecules are able to interact with the gRNA in RNP2. However, an unblocked primer molecule has a substantially higher likelihood than a blocked primer molecule to hybridize with the gRNA of RNP2.

FIG. 3A at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (309) comprise a quencher (310) and a fluorophore (311). As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (305) and RNP2 (308). The intact reporter moieties (309) become activated reporter moieties (312) when the quencher (310) is separated from the fluorophore (311), and the fluorophore emits a fluorescent signal (313). Signal strength increases rapidly as more blocked primer molecules (334) become unblocked primer molecules (344) generating synthesized activating molecules (346) and triggering activation of more RNP2 (308) complexes and more trans-cleavage activity of the reporter moieties (309). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4 . Also, as with the cascade assay embodiment utilizing blocked nucleic acid molecules that are not blocked primers, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected.

FIG. 3B is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and circular template molecules. The cascade assay of FIG. 3B differs from that depicted in FIG. 3A by the configuration of the template molecule. Where the template molecule in FIG. 3A was linear, in FIG. 3B the template molecule is circular. At left of FIG. 3B is a cascade assay reaction mix comprising 1) RNP1s (301) (only one RNP1 is shown); 2) RNP2s (302); 3) a circular template molecule (352) (non-target strand); 4) a circular blocked primer molecule (334); and 5) a polymerase (338), such as a (D29 polymerase. The circular template molecule (352) (non-target strand) comprises a PAM sequence (331) and a primer binding domain (PBD) (332). Blocked primer molecule (334) comprises a cleavable region (335) and a complement to the PBD (332) on the circular template molecule (352).

Upon addition of a sample comprising a target nucleic acid of interest (304) (capable of complexing with the gRNA in RNP1 (301)), the target nucleic acid of interest (304) binds to and activates RNP1 (305) but does not interact with or activate RNP2 (302). Once activated, RNP1 cuts the target nucleic acid of interest (304) via sequence specific cis-cleavage, which activates non-specific trans-cleavage of other nucleic acids present in the reaction mix, including at least one of the blocked primer molecules (334). The circular blocked primer molecule (334), upon cleavage, becomes an unblocked linear primer molecule (344), which has a region (336) complementary to the PBD (332) on the circular template molecule (352) and can hybridize with the circular template molecule (352).

Once the unblocked linear primer molecule (344) and the circular template molecule (352) are hybridized (i.e., hybridized at the PBD (332) of the circular template molecule (352) and the PBD complement (336) on the unblocked linear primer molecule (344)), 3′→5′ exonuclease activity of the polymerase (338) removes the unhybridized single-stranded DNA at the 3′ end of the unblocked primer molecule (344). The polymerase (338) can now use the circular template molecule (352) (non-target strand) to produce concatenated activating nucleic acid molecules (360) (which are concatenated target strands), which will be cleaved by the trans-cleavage activity of activated RNP1. The cleaved regions of the concatenated synthesized activating molecules (360) (target strand) are capable of activating the RNP2 (302→308) complex.

As described above, because the nucleic acid-guided nuclease in RNP2 (308) comprises both cis- and trans-cleavage activity, more blocked primer molecules (334) become unblocked primer molecules (344) triggering activation of more RNP2s (308) and more trans-cleavage activity in a cascade. FIG. 3B at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (309) comprise a quencher (310) and a fluorophore (311). As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (305) and RNP2 (308). The intact reporter moieties (309) become activated reporter moieties (312) when the quencher (310) is separated from the fluorophore (311), and the fluorescent signal (313) is unquenched and can be detected. Signal strength increases rapidly as more blocked primer molecules (334) become unblocked primer molecules (344) generating synthesized activating nucleic acid molecules and triggering activation of more RNP2s (308) and more trans-cleavage activity of the reporter moieties (309). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4 . Also note that as with the other embodiments of the cascade assay, in this embodiment, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected.

The polymerases used in the “blocked primer molecule” embodiments serve to polymerize a reverse complement strand of the template molecule (non-target strand) to generate a synthesized activating molecule (target strand) as described above. In some embodiments, the polymerase is a DNA polymerase, such as a BST, T4, or Therminator polymerase (New England BioLabs Inc., Ipswich MA., USA). In some embodiments, the polymerase is a Klenow fragment of a DNA polymerase. In some embodiments the polymerase is a DNA polymerase with 5′→3′ DNA polymerase activity and 3′→5′ exonuclease activity, such as a Type I, Type II, or Type III DNA polymerase. In some embodiments, the DNA polymerase, including the Phi29, T7, Q5®, Q5U®, Phusion®, OneTaq®, LongAmp®, Vent®, or Deep Vent® DNA polymerases (New England BioLabs Inc., Ipswich MA., USA), or any active portion or variant thereof. Also, a 3′ to 5′ exonuclease can be separately used if the polymerase lacks this activity.

FIG. 4 depicts three mechanisms in which a cascade assay reaction can release a signal from a reporter moiety. FIG. 4 at top shows the mechanism discussed in relation to FIGS. 2A, 3A and 3B. In this embodiment, a reporter moiety 409 is a separate molecule from the blocked nucleic acid molecules present in the reaction mix. Reporter moiety (409) comprises a quencher (410) and a fluorophore (411). An activated reporter moiety (412) emits a signal from the fluorophore (411) once it has been physically separated from the quencher (410).

Reporter Moiety Configurations

FIG. 4 at center shows a blocked nucleic acid molecule (403), which is also a reporter moiety. In addition to quencher (410) and fluorophore (411), a blocking moiety (407) can be seen (see also blocked nucleic acid molecules 203 in FIG. 2A). Blocked nucleic acid molecule/reporter moiety (403) comprises a quencher (410) and a fluorophore (411). In this embodiment of the cascade assay, when the blocked nucleic acid molecule (403) is unblocked due to trans-cleavage initiated by the target nucleic acid of interest binding to RNP1, the unblocked nucleic acid molecule (406) also becomes an activated reporter moiety with fluorophore (411) separated from quencher (410). Note both the blocking moiety (407) and the quencher (410) are removed. In this embodiment, reporter signal is directly generated as the blocked nucleic acid molecules become unblocked. Embodiments of this schema can be used to supply the bulky modifications to the blocked nucleic acid molecules described below.

FIG. 4 at the bottom shows that cis-cleavage of an unblocked nucleic acid molecule or a synthesized activating molecule at a PAM distal sequence by RNP2 generates a signal. Shown are activated RNP2 (408), unblocked nucleic acid molecule (461), quencher (410), and fluorophore (411) forming an activated RNP2 with the unblocked nucleic acid/reporter moiety intact (460). Cis-cleavage of the unblocked nucleic acid/reporter moiety (461) results in an activated RNP2 with the reporter moiety activated (462), comprising the activated RNP2 (408), the unblocked nucleic acid molecule with the reporter moiety activated (463), quencher (410) and fluorophore (411). Embodiments of this schema also can be used to supply the bulky modifications to the blocked nucleic acid molecules described below, and in fact a combination of the configurations of reporter moieties shown in FIG. 4 at center and at bottom may be used.

Preventing Undesired Blocked Nucleic Acid Molecule Unwinding

The present disclosure improves upon the signal cascade assay described in U.S. Ser. Nos. 17/861,207; 17/861,208; and 17/861,209 by addressing the problem with undesired “unwinding” of the blocked nucleic acid molecule. As described above in detail in relation to FIGS. 1B, 2A, 2B, 2G, 3A, 3B, and 4 , the cascade assay is initiated when a target nucleic acid of interest binds to and activates a first pre-assembled ribonucleoprotein complex (RNP1). The gRNA of RNP1 (gRNA1), comprising a sequence complementary to the target nucleic acid of interest, guides RNP1 to the target nucleic acid of interest. Upon binding of the target nucleic acid of interest to RNP1, RNP1 becomes activated, and the target nucleic acid of interest is cleaved in a sequence specific manner (i.e., cis-cleavage) while also triggering non-sequence specific, indiscriminate trans-cleavage activity which unblocks the blocked nucleic acid molecules in the reaction mix. The unblocked nucleic acid molecules can then activate a second pre-assembled ribonucleoprotein complex (RNP2), where RNP2 comprises a second gRNA (gRNA2) comprising a sequence complementary to the unblocked nucleic acid molecules, and at least one of the unblocked nucleic acid molecules is cis-cleaved in a sequence specific manner. Binding of the unblocked nucleic acid molecule to RNP2 leads to cis-cleavage of the unblocked nucleic acid molecule and non-sequence specific, indiscriminate trans-cleavage activity by RNP2, which in turn unblocks more blocked nucleic acid molecules (and reporter moieties) in the reaction mix activating more RNP2s. Each newly activated RNP2 activates more RNP2s, which in turn cleave more blocked nucleic acid molecules and reporter moieties in a reaction cascade, where all or most of the signal generated comes from the trans-cleavage activity of RNP2.

The improvement to the signal boost cascade assay described herein is drawn to preventing undesired unwinding of the blocked nucleic acid molecules in the reaction mix before the blocked nucleic acid molecules are unblocked via trans-cleavage; that is, preventing undesired unwinding that happens not as a result of unblocking due to trans-cleavage subsequent to cis-cleavage of the target nucleic acid of interest or trans-cleavage of unblocked nucleic acid molecules, but due to other factors. For a description of undesired unwinding, please see FIG. 1C and the attendant description herein. Minimizing undesired unwinding serves two purposes. First, preventing undesired unwinding that happens not as a result of designed or engineered unblocking leads to a “leaky” cascade assay system, which in turn leads to non-specific signal generation and false positives.

Second, preventing undesired unwinding limits non-specific interactions between the nucleic acid-guided nucleases (here, the RNP2s) and blocked nucleic acid molecules (i.e., the target nucleic acids for RNP2) such that only blocked nucleic acid molecules that become unblocked due to trans-cleavage activity react with the nucleic acid-guided nucleases. This “fidelity” in the cascade assay leads primarily to desired interactions and limits “wasteful” interactions where the nucleic acid-guided nucleases are essentially interacting with blocked nucleic acid molecules rather than interacting with unblocked nucleic acid molecules. That is, if unwinding is minimized the nucleic acid-guided nucleases are focused on desired interactions which then leads to immediate signal generation in the cascade assay. Preventing undesired unwinding leads to a more efficient cascade assay system providing more accurate quantification yet with the rapid results characteristic of the cascade assay (see FIGS. 10A-10H and 12 below).

Ratio of RNP2 to Blocked Nucleic Acid Molecules or Blocked Primers

In one modality to prevent undesired unwinding, the present disclosure describes using an unconventional ratio of blocked nucleic acid molecule (i.e., the target molecule for RNP2) and an RNP complex, here RNP2. The unconventional ratio may be used along with the blocked nucleic acid molecules and RNP2s described above as a primary method for minimizing unwinding or may be used in combination with the other modalities described below to minimize unwinding even more. For example, if one were to design an ideal blocked nucleic acid molecule having an “infinite K_(d)” such as, e.g., through design of the blocked nucleic acid molecule (or blocked primer molecule) and/or inclusion of bulky modifications on the blocked nucleic acid molecule (or blocked primer molecule), the ratio of blocked nucleic acid molecules to RNP2s would not affect the reaction mix to any discernable degree. The common wisdom of the ratio of enzyme to target (here, RNP2 to blocked nucleic acid molecule) is that results are achieved—a signal is generated—when there is a high concentration of nucleic acid-guided nuclease (i.e., RNP complex) and a lower concentration of target or, stated another way, when there is a significant excess of nucleic acid-guided nuclease to target. As described above, in CRISPR detection/diagnostic assay protocols known to date, the CRISPR enzyme (i.e., nucleic acid-guided nuclease) is far in excess of blocked nucleic acid molecules (see, Sun, et al., J. of Translational Medicine, 12:74 (2021); Broughton, et al., Nat. Biotech., 38:870-74 (2020); and Lee, et al., PNAS, 117(41):25722-31 (2020)). However, in a cascade assay system where the nucleic acid-guided nuclease (or RNP complex) is in excess of the targets (here, the blocked nucleic acid molecules), the nucleic acid-guided nucleases encounter the blocked nucleic acid molecules repeatedly, probing the blocked nucleic acid molecules and subjecting them to unwinding. If the blocked nucleic acid molecules are probed and unwound repeatedly, they finally unwind which then triggers activation of RNP2 and cis-cleavage of the blocked nucleic acid molecule even in the absence of a target nucleic acid of interest and the trans-cleavage activity generated thereby.

However, by adjusting the ratio of RNP2 to blocked nucleic acid molecules such that there is an excess of blocked nucleic acid molecules to RNP2, any one blocked nucleic acid molecule may be probed by RNP2; however, the likelihood that any one blocked nucleic acid molecule will be probed repeatedly (and thus unwound) is much lower. If a blocked nucleic acid molecule is probed but then has time to re-hybridize or “recover”, that blocked nucleic acid molecule will stay blocked, will not be subject to non-specific unwinding, and will not trigger activation of RNP2. That is, how often any one blocked nucleic acid molecule is probed is important. As long as an improperly probed blocked nucleic acid has time to re-hybridize after unwinding, there is far less chance that the blocked nucleic acid will be unblocked (i.e., unwound) and will trigger signal generation. That is, preventing non-specific unwinding of the blocked nucleic acid molecules makes the nucleic acid-guided nuclease available for desired unwinding interactions.

In order to prevent non-specific unwinding as described herein, the ratio of blocked nucleic acid molecules to RNP2 should be about 50:1, or about 40:1, or about 35:1, or about 30:1, or about 25:1, or about 20:1, or about 15:1, or about 10:1, or about 7.5:1, or about 5:1, or about 4:1, or about 3:1, or about 2.5:1, or about 2:1, or about 1.5:1, or at least where the molar concentration of blocked nucleic acid molecules is equal to or greater than the molar concentration of RNP2s. As noted above, the signal amplification cascade assay reaction mixture typically contains about 1 fM to about 1 mM of a given RNP2, or about 1 pM to about 500 μM of a given RNP2, or about 10 pM to about 100 μM of a given RNP2; thus, the signal amplification cascade assay reaction mixture typically contains about 2.5 fM to about 2.5 mM blocked nucleic acid molecules, or about 2.5 pM to about 1.25 mM blocked nucleic acid molecules, or about 25 pM to about 250 μM blocked nucleic acid molecules. That is, the reaction mixture contains about 6×10⁴ to about 6×10¹⁴ RNP2s per microliter (μl) or about 6×10⁶ to about 6×10¹² RNP2s per microliter (μl) and thus about 6×10⁴ to about 6×10¹⁴ RNP2s per microliter (μl) or about 6×10⁶ to about 6×10¹² blocked nucleic acid molecules per microliter (μl). Note, the ratios may be used along with the blocked nucleic acid molecules and RNP2s described above as a primary method for minimizing unwinding or the ratios of blocked nucleic acid molecules to RNP2s may be used in combination with the other modalities described below to further minimize unwinding. Again, if one were to design an ideal blocked nucleic acid molecule having an “infinite K_(d)”, the ratio of blocked nucleic acid molecules to RNP2s would not affect the reaction mix to any discernable degree and the ratios of blocked nucleic acid molecules to RNP2s would not necessarily be within these ranges.

Variant Engineered Nucleic Acid-Guided Nucleases

In some embodiments, the protein sequence of the Cas12a nucleic acid-guided nuclease is modified, with e.g., mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules (see Shin et al., Front. Genet., 11:1577 (2021); doi: 10.3389/fgene.2020.571591, herein incorporated by reference; and Yamano et al., Mol. Cell, 67(4): 633-645 (2017); doi: 10.1016/j.molcel.2017.06.035, herein incorporated by reference) such that the variant engineered nucleic acid-guided nuclease has reduced (or absent) PAM specificity, relative to the unmodified or wildtype nucleic acid-guided nuclease and reduced cleavage activity in relation to double strand DNA with or without a PAM. Such enzymes are referred to herein as single-strand-specific Cas12a nucleic acid-guided nucleases or variant engineered nucleic acid-guided nucleases.

FIG. 5 is a simplified block diagram of an exemplary method 500 for designing, synthesizing and screening variant nucleic acid-guided nucleases. In a first step, mutations or modifications to a nucleic acid-guided nuclease are designed 502, based on, e.g., homology to related nucleic acid-guided nucleases, predicted protein structure and active site configuration, and mutagenesis modeling. For assessment of homologies to other nucleic acid-guided nucleases, amino acid sequences may be found in publicly available databases known to those with skill in the art, including, e.g., Protein DataBank Europe (PDBe), Protein Databank Japan (PDBj), SWISS-PROT, GenBank, RefSeq, TrEMBL, PROSITE, DisProt, InterPro, PIR-International, and PRF/SEQDB. Amino acid homology alignments for purposes of determining similarities to known nucleic acid-guided nucleases can be performed using CUSTALW, CUSTAL OMEGA, COBALT: Multiple Alignment Tool; SIM; and PROBCONS.

For protein engineering and amino acid substitution model predictions for each of the desired mutations, protein modeling software such as SWISS-MODEL, HHpred, I-TASSER, IntFOLD, RaptorX, FoldX, Rosetta, and trRosetta may be used to simulate the structural change(s) and to calculate various parameters due to the structural changes as a result of the amino acid substitution(s), including root mean square deviation (RMSD) value in Angstrom units (i.e., a measurement of the difference between the backbones of the initial nucleic acid-guided nuclease and the mutated nucleic acid nucleic acid-guided nuclease) and changes to the number of hydrogen bonds and conformation in the active site. For the methods used to generate the variant engineered nucleic acid-guided nucleases described herein, see Example VII below.

Following modelling, coding sequences for the variant nucleic acid-guided nucleases that appear to deliver desired properties are synthesized and inserted into an expression vector 504. Methods for site-directed mutagenesis are known in the art, including PCR-based methods such as traditional PCR, where primers are designed to include the desired change; primer extension, involving incorporating mutagenic primers in independent nested PCR before combining them in the final product; and inverse PCR. Additionally, CRISPR gene editing may be performed to introduce the desired mutation or modification to the nucleic acid-guided nuclease coding sequence. The mutated (variant) coding sequences are inserted into an expression vector backbone comprising regulatory sequences such as enhancer and promoter regions. The type of expression vector (e.g., plasmid or viral vector) will vary depending on the type of cells to be transformed.

At step 506, cells of choice are transformed with the variant expression vectors. A variety of delivery systems may be used to introduce (e.g., transform or transfect) the expression vectors into a host cell, including the use of yeast systems, lipofection systems, microinjection systems, biolistic systems, virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acid conjugates, virions, artificial virions, viral vectors, electroporation, cell permeable peptides, nanoparticles, nanowires, exosomes. Once cells are transformed (or transfected), the transformants are allowed to recover and grow.

Following transformation, the cells are screened for expression of nucleic acid-guided nucleases with desired properties 508, such as cut activity or lack thereof, paste activity or lack thereof, PAM recognition or changes thereto, stability and the ability to form RNPs at various temperatures, and/or cis- and trans-cleavage activity at various temperatures. The assays used to screen the variant nucleic acid-guided nucleases will vary depending on the desired properties, but may include in vitro and in vivo PAM depletion, assays for editing efficiency such as a GFP to BFP assay, and, as used to assess the variant nucleic acid-guided nucleases described herein, in vitro transcription/translation (IVTT) assays were used to measure in vitro trans cleavage with both dsDNA and ssDNA and with and without the presence of a PAM in the blocked nucleic acid molecules, where dsDNA should not activate trans-cleavage regardless of the presence of PAM sequence.

After screening the variant nucleic acid-guided nucleases via the IVTT assays, variants with the preferred properties are identified and selected 510. At this point, a variant may be chosen 512 to go forward into production for use in, e.g., the CRISPR cascade systems described herein; alternatively, promising mutations and/or modifications may be combined 514 and the construction, screening and identifying process is repeated.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease may not recognize one or more of the following PAM or partial PAM sequences (listed from 5′ to 3′): TTTN, TTTV, CTTA, CTTV, TCTV, TTCV, YTV, or YTN wherein “A” represents adenine, “C” represents cytosine, “T” represents thymine, “G” represents guanine, “V” represents guanine or cytosine or adenine, “Y” represents guanine or adenine, and “N” represents any nucleotide. In some embodiments, the Cas12a nucleic acid-guided nuclease may have reduced recognition for one or more of the following PAM or partial PAM sequences (listed from 5′ to 3′): TTTN, TTTV, CTTA, CTTV, TCTV, TTCV, YTV, or YTN. The single-strand-specific Cas12a nucleic acid-guided nucleases described herein may have at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, such as about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) reduced recognition (i.e., specificity) for one or more of the following PAM or partial PAM sequences (listed from 5′ to 3′): TTTN, TTTV, CTTA, CTTV, TCTV, TTCV, YTV, or YTN.

Exemplary wild type (WT) Cas12a protein sequences are described in Table 7 below. FIG. 6A shows the result of protein structure prediction using Rosetta and SWISS modeling of wildtype LbCas 12a (Lachnospriaceae bacterium Cas 12a), and FIG. 6B shows the result of example mutations on the LbCas 12a protein structure prediction using Rosetta and SWISS modeling of LbCas12a and indicating the PAM regions (described in more detail in relation to Example VII). Any of these sequences (e.g., SEQ ID NOs: 1-15 and homologs or orthologs thereof) may be modified, as described herein, to generate a single-strand-specific nucleic acid-guided nuclease.

TABLE 7 Exemplary wild type Cas12a nucleic acid-guided nucleases Species SEQ Name ID Reference ID NO: Protein Sequence Lachnospiraceae SEQ MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAED bacterium Cas12a ID YKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENK (LbCas12a) NO: 1 ELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIAL PDD: 6KL9_A VNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNM DIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGI DVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQV LSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKN FDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKK KAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQ KVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKS FENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYV TQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYL AIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSK KWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISR YPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEV DKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHG QIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLS YDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPY VIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSL LDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAV IALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPC ATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVN LLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADY IKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGI NYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDF LISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAI GQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH Acidaminococcus SEQ MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH sp. Cas12a ID YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETR (AsCas12a) NO: 2 NALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFN NCBI Ref.: GKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDI WP_021736722.1 STAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFV STSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVI QSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSAL CDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIIS AAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQL DSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA RNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNG LYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIP KCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKK FQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRP SSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRP KSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLS HDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAA NSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRS LNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVI HEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLID KLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVP APYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGD FILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGK RIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLEN DDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDS RFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISN QDWLAYIQELRN Candidatus SEQ MNNYDEFTKLYPIQKTIRFELKPQGRTMEHLETFNFFEEDRDRAEK Methanoplasma ID YKILKEAIDEYHKKFIDEHLTNMSLDWNSLKQISEKYYKSREEKDK termitum NO: 3 KVFLSEQKRMRQEIVSEFKKDDRFKDLFSKKLFSELLKEEIYKKGN (CtCas12a) HQEIDALKSFDKFSGYFIGLHENRKNMYSDGDEITAISNRIVNENFP NCBI Gene ID: KFLDNLQKYQEARKKYPEWIIKAESALVAHNIKMDEVFSLEYFNK 24818655 VLNQEGIQRYNLALGGYVTKSGEKMMGLNDALNLAHQSEKSSKG RIHMTPLFKQILSEKESFSYIPDVFTEDSQLLPSIGGFFAQIENDKDG NIFDRALELISSYAEYDTERIYIRQADINRVSNVIFGEWGTLGGLMR EYKADSINDINLERTCKKVDKWLDSKEFALSDVLEAIKRTGNNDA FNEYISKMRTAREKIDAARKEMKFISEKISGDEESIHIIKTLLDSVQQ FLHFFNLFKARQDIPLDGAFYAEFDEVHSKLFAIVPLYNKVRNYLT KNNLNTKKIKLNFKNPTLANGWDQNKVYDYASLIFLRDGNYYLGI INPKRKKNIKFEQGSGNGPFYRKMVYKQIPGPNKNLPRVFLTSTKG KKEYKPSKEIIEGYEADKHIRGDKFDLDFCHKLIDFFKESIEKHKDW SKFNFYFSPTESYGDISEFYLDVEKQGYRMHFENISAETIDEYVEKG DLFLFQIYNKDFVKAATGKKDMHTIYWNAAFSPENLQDVVVKLN GEAELFYRDKSDIKEIVHREGEILVNRTYNGRTPVPDKIHKKLTDY HNGRTKDLGEAKEYLDKVRYFKAHYDITKDRRYLNDKIYFHVPLT LNFKANGKKNLNKMVIEKFLSDEKAHIIGIDRGERNLLYYSIIDRSG KIIDQQSLNVIDGFDYREKLNQREIEMKDARQSWNAIGKIKDLKEG YLSKAVHEITKMAIQYNAIVVMEELNYGFKRGRFKVEKQIYQKFE NMLIDKMNYLVFKDAPDESPGGVLNAYQLTNPLESFAKLGKQTGI LFYVPAAYTSKIDPTTGFVNLFNTSSKTNAQERKEFLQKFESISYSA KDGGIFAFAFDYRKFGTSKTDHKNVWTAYTNGERMRYIKEKKRN ELFDPSKEIKEALTSSGIKYDGGQNILPDILRSNNNGLIYTMYSSFIA AIQMRVYDGKEDYIISPIKNSKGEFFRTDPKRRELPIDADANGAYNI ALRGELTMRAIAEKFDPDSEKMAKLELKHKDWFEFMQTRGD Eubacterium SEQ MNGNRSIVYREFVGVIPVAKTLRNELRPVGHTQEHIIQNGLIQEDEL eligens ID RQEKSTELKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSSP (EeCas12a) NO: 4 SKDNKKALEKEQSKMREQICTHLQSDSNYKNIFNAKLLKEILPDFI NCBI Gene ID: KNYNQYDVKDKAGKLETLALFNGFSTYFTDFFEKRKNVFTKEAVS 41356122 TSIAYRIVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGD WELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTKNN YNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETE KGNIIGKLKDIVNKYDELDEKRIYISKDFYETLSCFMSGNWNLITGC VENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDE KERNEFKNSNAKQYIREISNIITDTETAHLEYDDHISLIESEEKADEM KKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVP LYNRVRNYVTQKPYNSKKIKLNFQSPTLANGWSQSKEFDNNAIILI RDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGA NKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLI DYFKNSIEKHAEWRKYEFKFSATDSYSDISEFYREVEMQGYRIDW TYISEADINKLDEEGKIYLFQIYNKDFAENSTGKENLHTMYFKNIFS EENLKDIIIKLNGQAELFYRRASVKNPVKHKKDSVLVNKTYKNQL DNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRT AQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMVVKYIAQN DDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVE KEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLIVEYNAIIAM EDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKEKSVDEPGG LLKGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAFNF KSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITMGK TQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLEDNEINYA DGHDIRIDMEKMDEDKKSEFFAQLLSLYKLTVQMRNSYTEAEEQE NGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANGAYC IALKGLYEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRYE Moraxella SEQ MLFQDFTHLYPLSKTVRFELKPIGKTLEHIHAKNFLNQDETMADM bovoculi Cas12a ID YQKVKAILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKD (Mb3Cas12a) NO: 5 DGLQKQLKDLQAVLRKEIVKPIGNGGKYKAGYDRLFGAKLFKDG GenBank: KELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMY AKG12737.1 SDEDKHTAIAYRLIHENLPRFIDNLQILATIKQKHSALYDQIINELTA SGLDVSLASHLDGYHKLLTQEGITAYNTLLGGISGEAGSRKIQGINE LINSHHNQHCHKSERIAKLRPLHKQILSDGMGVSFLPSKFADDSEV CQAVNEFYRHYADVFAKVQSLFDGFDDYQKDGIYVEYKNLNELS KQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTK EKDKFIKGVHSLASLEQAIEHYTARHDDESVQAGKLGQYFKHGLA GVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSDKSPEIRQLKEL LDNALNVAHFAKLLTTKTTLHNQDGNFYGEFGALYDELAKIATLY NKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQK DGCYYLALLDKAHKKVFDNAPNTGKSVYQKMIYKLLPGPNKMLP KVFFAKSNLDYYNPSAELLDKYAQGTHKKGDNFNLKDCHALIDFF KAGINKHPEWQHFGFKFSPTSSYQDLSDFYREVEPQGYQVKFVDIN ADYINELVEQGQLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDN LVNPIYKLNGEAEIFYRKASLDMNETTIHRAGEVLENKNPDNPKKR QFVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSI QQYDEVNVIGIDRGERHLLYLTVINSKGEILEQRSLNDITTASANGT QMTTPYHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQIS QLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHL VLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTS KIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNADRGYFEFHIDY AKFNDKAKNSRQIWKICSHGDKRYVYDKTANQNKGATIGVNVND ELKSLFTRYHINDKQPNLVMDICQNNDKEFHKSLMYLLKTLLALR YSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIA LKGLWLLNELKNSDDLNKVKLAIDNQTWLNFAQNR Francisella SEQ MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDY novicida Cas12a ID KKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNL (FnCas12a) NO: 6 QKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL UniProtKB/Swiss- WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRK Prot: A0Q7Q2.1 NVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFN TIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQIL SDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSL LFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYIT QQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDI DKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQ ASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFY LVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANG WDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGE GYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTK NGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQR YNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDF SAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIP KKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPIT INFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDG KGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNI KEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQ VYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKK MGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFD KICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDK NHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFA KLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMP QDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQ NRNN Francisella SEQ MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDY tularensis subsp. ID KKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNL novicida FTG NO: 7 QKDFKSAKDTIKKQISKYINDSEKFKNLFNQNLIDAKKGQESDLIL Cas12a WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRK (FnoCas12a) NVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK NCBI Gene ID: KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFN 60806594 TIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQIL SDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSL LFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYIT QQVAPKNLDNPSKKEQDLIAKKTEKAKYLSLETIKLALEEFNKHRD IDKQCRFEEILSNFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLL QASAEEDVKAIKDLLDQTNNLLHRLKIFHISQSEDKANILDKDEHF YLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLASG WDKNKESANTAILFIKDDKYYLGIMDKKHNKIFSDKAIEENKGEG YKKIVYKQIADASKDIQNLMIIDGKTVCKKGRKDRNGVNRQLLSL KRKHLPENIYRIKETKSYLKNEARFSRKDLYDFIDYYKDRLDYYDF EFELKPSNEYSDFNDFTNHIGSQGYKLTFENISQDYINSLVNEGKLY LFQIYSKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAE LFYRKQSIPKKITHPAKETIANKNKDNPKKESVFEYDLIKDKRFTED KFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLA YYTLVDGKGNIIKQDNFNIIGNDRMKTNYHDKLAAIEKDRDSARK DWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRG RFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTA PFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKS QEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRL INFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICG ESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDS RQAPKNMPQDADANGAYHIGLKGLMLLDRIKNNQEGKKLNLVIK NEEYFEFVQNRNN Flavobacteriales SEQ MKNNNMLNFTNKYQLSKTLRFELKPIGKTKENIIAKNILKKDEERA bacterium ID ESYQLMKKTIDGFHKHFIELAMQEVQKTKLSELEEFAELYNKSAEE (FbCas12a) NO: 8 KKKDDKFDDKFKKVQEALRKEIVKGFNSEKVKYYYSNIDKKILFT NCBI Gene ID: ELLKNWIPNEKMITELSEWNAKTKEEKEHLVYLDKEFENFTTYFG MBE7442138.1 GFHKNRENMYTDKEQSTAIAYRLIHENLPKFLDNINIYKKVKEIPV LREECKVLYKEIEEYLNVNSIDEVFELSYYNKTLTQKDIDVYNLIIG GRTLEEGKKKIQGLNEYINLYNQKQEKKNRIPKLKILYKQILSDRDS ISWLPESFEDDNEKTASQKVLEAINLYYRDNLLCFQPKDKKDTENV LEETKKLLAGLSTSDLSKIYIRNDRAITDISQALFKDYGVIKDALKF QFIQSFTIGKNGLSKKQEEAIEKHLKQKYFSIAEIENALFTYQSETDA LKELKENSHPVVDYFINHFKAKKKEETDKDFDLIANIDAKYSCIKG LLNTPYPKDKKLYQRSKGDNDIDNIKAFLDALMELLHFVKPLALS NDSTLEKDQNFYSHFEPYYEQLELLIPLYNKVRNFAAKKPYSTEKF KLNFDNATLLNGWDKNKETDNTSVILRKDGLYYLAIMPQDNKNV FKDSPDLKANENCFEKMDYKQMALPMGFGAFVRKCFGTASQLG WNCPESCKNEEDKIIIKEDEVKNNRAEIIDCYKDFLNIYEKDGFQYK EYGFDFKESNKYESLREFFIDVEQQGYKITFQNISENYINQLVEDGK LYLFQIYNKDFSPYSKGKPNMHTMYWKALFDSENLKDVVYKLNG QAEVFYRKKSIEQKNIVTHKANEPIDNKNPKAKKKQSTFEYDLIKD KRYTVDKFQFHVPITLNFKATGNDYINQDVLTYLKNNPEVNIIGLD RGERHLIYLTLINQKGEILLQESLNTIVNKKYDIETPYHTLLQNKED ERAKARENWGVIENIKELKEGYISQVVHKIAKLMVEYNAIVVMED LNTGFKRGRFKVEKQVYQKLEKMLIDKLNYLVFKDKDPSEVGGL YHALQLTNKFENFSKIGKQSGFLFYVPAWNTSKIDPTTGFVNLFNT KYESVPKAQEFFKKFKSIKFNSAENYFEFAFDYNDFTTRAEGTKTD WIVCTYGDRIKTFRNPDKVNQWDNQEVNLTEQFEDFFGKNNLIYG DGNCIKNQIILHDKKEFFEGLLHLLKLTLQMRNSITNSEVDYLISPV KNNKGEFYDSRKANNTLPKDADANGAYHIAKKGLVLLNRLKENE VEEFEKSKKVKDGKSQWLPNKDWLDFVQRNVEDMVVV Lachnospira SEQ MNGNRSIVYREFVGVTPVAKTLRNELRPVGHTQEHIIQNGLIQEDE eligens ID LRQEKSTELKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSS (Lb4Cas12a) NO: 9 PSKDNKKALEKEQSKMREQICTHLQSDSNYKNIFNAKLFKEILPDFI NCBI Gene ID: KNYNQYDVKDKAGKLETVALFNGFSTYFTDFFEKRKNVFTKEAV MBS6299380.1 STSIAYRIVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGD WELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTRNN YNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETE KGNIIVKLKDIVNKYDELDEKRIYISKDFYETLSCFISGNWNLITGC VENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDE KERNEFKNSNAKQYIREISNIITDTETAHLEYDEHISLIESEEKADEM KKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVP LYNRVRNYVTQKPYNSKKIKLNFQSPTLANGWSQSKEFDNNAIILI RDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGA NKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLI DYFKNSIEKHAEWRKYEFKFSATDSYNDISEFYREVEMQGYRIDW TYISEADINKLDEEGKIYLFQIYNKYFAENSTGKENLHTMYFKNIFS EENLKDIIIKLNGQAELFYRRASVKNPVKHKKDSVLVNKTYKNQL DNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRT AQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMAVKYIAQN DDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVE KEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLMVEYNAIIA MEDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKGKSVDEP GGLLRGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAF NFKSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITM GKTQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLKDNKIN YADGHDVRIDMEKMDEDKNSEFFAQLLSLYKLTVQMRNSYTEAE EQEKGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANG AYCIALKGLYEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRY E Moraxella SEQ MLFQDFTHLYPLSKTVRFELKPIGRTLEHIHAKNFLSQDETMADMY bovoculi ID QKVKVILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDD (MbCas12a) NO: GLQKQLKDLQAVLRKESVKPIGSGGKYKTGYDRLFGAKLFKDGK NCBI Gene ID: 10 ELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMYS WP_046697655.1 DEDKHTAIAYRLIHENLPRFIDNLQILTTIKQKHSALYDQIINELTAS GLDVSLASHLDGYHKLLTQEGITAYNRIIGEVNGYTNKHNQICHKS ERIAKLRPLHKQILSDGMGVSFLPSKFADDSEMCQAVNEFYRHYT DVFAKVQSLFDGFDDHQKDGIYVEHKNLNELSKQAFGDFALLGR VLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSL ASLEQAIEHHTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNN HSTIKGFLERERPAGERALPKIKSGKNPEMTQLRQLKELLDNALNV AHFAKLLTTKTTLDNQDGNFYGEFGVLYDELAKIPTLYNKVRDYL SQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDGCYYLA LLDKAHKKVFDNAPNTGKNVYQKMVYKLLPGPNKMLPKVFFAK SNLDYYNPSAELLDKYAKGTHKKGDNFNLKDCHALIDFFKAGINK HPEWQHFGFKFSPTSSYRDLSDFYREVEPQGYQVKFVDINADYIDE LVEQGKLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDNLADPIY KLNGEAQIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYD IIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDE VNVIGIDRGERHLLYLTVINSKGEILEQRSLNDITTASANGTQVTTP YHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQINQLMLK YNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDK ADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPET GFVDLLKPRYENIAQSQAFFGKFDKICYNTDKGYFEFHIDYAKFTD KAKNSRQKWAICSHGDKRYVYDKTANQNKGAAKGINVNDELKS LFARYHINDKQPNLVMDICQNNDKEFHKSLMCLLKTLLALRYSNA SSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGL WLLNELKNSDDLNKVKLAIDNQTWLNFAQNR Prevotella bryantii SEQ MKFTDFTGLYSLSKTLRFELKPIGKTLENIKKAGLLEQDQHRADSY (Pb2Cas12a) ID KKVKKIIDEYHKAFIEKSLSNFELKYQSEDKLDSLEEYLMYYSMKR NCBI Gene ID: NO: IEKTEKDKFAKIQDNLRKQIADHLKGDESYKTIFSKDLIRKNLPDFV WP_039871282.1 11 KSDEERTLIKEFKDFTTYFKGFYENRENMYSAEDKSTAISHRIIHEN LPKFVDNINAFSKIILIPELREKLNQIYQDFEEYLNVESIDEIFHLDYF SMVMTQKQIEVYNAIIGGKSTNDKKIQGLNEYINLYNQKHKDCKL PKLKLLFKQILSDRIAISWLPDNFKDDQEALDSIDTCYKNLLNDGN VLGEGNLKLLLENIDTYNLKGIFIRNDLQLTDISQKMYASWNVIQD AVILDLKKQVSRKKKESAEDYNDRLKKLYTSQESFSIQYLNDCLR AYGKTENIQDYFAKLGAVNNEHEQTINLFAQVRNAYTSVQAILTTP YPENANLAQDKETVALIKNLLDSLKRLQRFIKPLLGKGDESDKDER FYGDFTPLWETLNQITPLYNMVRNYMTRKPYSQEKIKLNFENSTLL GGWDLNKEHDNTAIILRKNGLYYLAIMKKSANKIFDKDKLDNSGD CYEKMVYKLLPGANKMLPKVFFSKSRIDEFKPSENIIENYKKGTHK KGANFNLADCHNLIDFFKSSISKHEDWSKFNFHFSDTSSYEDLSDF YREVEQQGYSISFCDVSVEYINKMVEKGDLYLFQIYNKDFSEFSKG TPNMHTLYWNSLFSKENLNNIIYKLNGQAEIFFRKKSLNYKRPTHP AHQAIKNKNKCNEKKESIFDYDLVKDKRYTVDKFQFHVPITMNFK STGNTNINQQVIDYLRTEDDTHIIGIDRGERHLLYLVVIDSHGKIVE QFTLNEIVNEYGGNIYRTNYHDLLDTREQNREKARESWQTIENIKE LKEGYISQVIHKITDLMQKYHAVVVLEDLNMGFMRGRQKVEKQV YQKFEEMLINKLNYLVNKKADQNSAGGLLHAYQLTSKFESFQKLG KQSGFLFYIPAWNTSKIDPVTGFVNLFDTRYESIDKAKAFFGKFDSI RYNADKDWFEFAFDYNNFTTKAEGTRTNWTICTYGSRIRTFRNQA KNSQWDNEEIDLTKAYKAFFAKHGINIYDNIKEAIAMETEKSFFED LLHLLKLTLQMRNSITGTTTDYLISPVHDSKGNFYDSRICDNSLPAN ADANGAYNIARKGLMLIQQIKDSTSSNRFKFSPITNKDWLIFAQEK PYLND Candidatus SEQ MENKNNQTQSIWSVFTKKYSLQKTLRFELKPVGETKKWLEENDIF Parcubacteria ID KKDLNIDKSYNQAKFYFDKLHQDFIKESLSVENGIRNIDFEKFAKIF bacterium NO: ESNKEKIVSLKKKNKEVKDKNKKNWDEISKLEKEIEGQRENLYKEI (PgCas12a) 12 RELFDKRAEKWKKEYQDKEIERGGKKEKIKFSSADLKQKGVNFLT NCBI Gene ID: AAGIINILKYKFPAEKDEEFRKEGYPSLFINDELNPGKKIYIFESFDK BCX15829.1 FTTYLSKFQQTRENLYKDDGTSTAVATRIVSNFERFLENKSLFEEK YKNKAKDVGLTKEEEKVFEINYYYDCLIQEGIDKYNKIIGEINRKT KEYRDKNKIDKKDLPLFLNLEKQILGEVKKERVFIEAKDEKTEEEV FIDRFQEFIKRNKIKIYGDEKEEIEGAKKFIEDFTSGIFENDYQSIYLK KNVINEIVNKWFSNPEEFLMKLTGVKSEEKIKLKKFTSLDEFKNAIL SLEGDIFKSRFYKNEVNPEAPLEKEEKSNNWENFLKIWRFEFESLFK DKVEKGEIKKDKNGEPIQIFWGYTDKLEKEAEKIKFYSAEKEQIKTI KNYCDAALRINRMMRYFNLSDKDRKDVPSGLSTEFYRLVDEYFN NFEFNKYYNGIRNFITKKPSDENKIKLNFESRSLLDGWDVSKEKDN LGLIFIKNNKYYLGVLRKENSKLFDYQITEKDNQKEKERKNNLKNE ILANDNEDFYLKMNYWQIADPAKDIFNLVLMPDNTVKRFTKLEEK NKHWPDEIKRIKEKGTYKREKVNREDLVKIINYFRKCALIYWKKF DLKLLPSEEYQTFKDFTDHIALQGYKINFDKIKASYIEKQLNDGNL YLFEVSNKDFYKYKKPDSRKNIHTLYWEHIFSKENLEEIKYPLIRLN GKAEIFYRDVLEMNEEMRKPVILERLNGAKQAKREDKPVYHYQR YLKPTYLFHCPITLNADKPSSSFKNFSSKLNHFIKDNLGKINIIGIDR GEKNLLYYCVINQNQEILDYGSLNKINLNKVNNVNYFDKLVEREK QRQLERQSWEPVAKIKDLKQGYISYVVRKICDLIINHNAIVVLEDLS RRFKQIRNGISERTVYQQFEKALIDKLNYLIFKDNRDVFSPGGVLN GYQLAAPFTSFKDIEKAKQTGVLFYTSAEYTSQTDPLTGFRKNIYIS NSASQEKIKELINKLKKFGWDDTEESYFIEYNQVDFAEKKKKPLSK DWTIWTKVPRVIRWKESKSSYWSYKKINLNEEFRDLLEKYGFEAQ SNDILSNLKKRIAENDKLLVEKKEFDGRLKNFYERFIFLFNIVLQVR NTYSLSVEIDKTEKKLKKIDYGIDFFASPVKPFFTTFGLREIGIEKDG KVVKDNAREEIASENLAEFKDRLKEYKPEEKFDADGVGAYNIARK GLIILEKIKNNPNKPDLSISKEEWDKFVQR Acidaminococcus SEQ MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH sp. ID YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETR (AaCas12a) NO: NALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFN NCBI Gene ID: 13 GKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDI WP_021736722.1 STAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFV STSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVI QSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSAL CDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIIS AAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQL DSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA RNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNG LYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIP KCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKK FQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRP SSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRP KSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLS HDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAA NSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRS LNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVI HEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLID KLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVP APYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGD FILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGK RIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLEN DDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDS RFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISN QDWLAYIQELRN Bacteroidetes SEQ MESPTTQLKKFTNLYQLSKTLRFELKPVGKTKEHIETKGILKKDEE bacterium ID RAVNYKLIKKIIDGFHKHFIELAMQQVKLSKLDELAELYNASAERK (BoCas12a) NO: KEESYKKELEQVQAALRKEIVKGFNIGEAKEIFSKIDKKELFTELLD NCBI Gene ID: 14 EWVKNLEEKKLVDDFKTFTTYFTGFHENRKNMYTDKAQSTAIAY PKP47250.1 RLVHENLPKFLDNTKIFKQIETKFEASKIEEIETKLEPIIQGTSLSEIFT LDYYNHALTQAGIDFINNIIGGYTEDEGKKKIQGLNEYINLYNQKQ EKKNRIPKLKILYKQILSDRDSISFLPDAFEDSQEVLNAIQNYYQTN LIDFKPKDKEETENVLEETKKLLTELFSNELSKIYIRNDKAITDISQA LFNDWGVFKSALEYKFIQDLELGTKELSKKQENEKEKYLKQAYFSI AEIENALFAYQNETDVLNEIKENSHPIADYFTKHFKAKKKVDTSTS SVEKDFDLIANIDAKYSCIKGILNTDYPKDKKLNQEKKTIDDLKVFL DSLMELLHFVKPLALPNDSILEKDENFYSHFESYYEQLELLIPLYNK VRNYAAKKPYSTEKFKLNFENATLLKGWDKNKEIDNTSVILRKRG LYYLAIMPQDNKNVFKKSPNLKNNESCFEKMDYKQMALPMGFGA FVRKCFGTAFQLGWNCPKSCINEEDKIIIKEDEVKNNRAEIIDCYKD FLNIYEKDGFQYKEYGFNFKESKEYESLREFFIDVEQKGYKIEFQNI SENYIHQLVNEGKLYLFQIYNKDFSSYSKGKPNMHTMYWKALFDP ENLKDVVYKLNGQAEVFYRKKSIEDKNIITHKANEPIENKNPKAKK TQSTFEYDLIKDKRYTVDKFHFHVPITINFKATGNNYINQQVLDHL KNNTDVNIIGLDRGERHLIYLTLINQKGEILLQESLNTIVNKKFDIET PYHTLLQNKEDERAKARENWGVIENIKELKEGYLSQVVHKIAKLM VDYNAIVVMEDLNTGFKRGRFKVEKQVYQKLEKMLIDKLNYLVF KDKDPNEVGGLYNALQLTNKFESFSKMGKQSGFLFYVPAWNTSKI DPTTGFVNLFYAKYESIPKAQDFFTKFKSIRYNSDENYFEFAFDYN DFTTRAEGTKSDWTVCTYGDRIKTFRNPEKNNQWDNQEVNLIEQF EAFFGKHNITYGDGNCIKKQLIEQDKKEFFEELFHLFKLTLQMRNSI TNSEIDYLISPVKNSKKEFYDSRKADSTLPKDADANGAYHIAKKGL MWLEKINSFKGSDWKKLDLDKTNKTWLNFVQETASEKHKKLQTV Candidatus SEQ MDAKEFTGQYPLSKTLRFELRPIGRTWDNLEASGYLAEDRHRAEC Methanomethylop ID YPRAKELLDDNHRAFLNRVLPQIDMDWHPIAEAFCKVHKNPGNK hilus alvus NO: ELAQDYNLQLSKRRKEISAYLQDADGYKGLFAKPALDEAMKIAKE Mx1201 15 NGNESDIEVLEAFNGFSVYFTGYHESRENIYSDEDMVSVAYRITED (CMaCas12a) NFPRFVSNALIFDKLNESHPDIISEVSGNLGVDDIGKYFDVSNYNNF NCBI Gene ID: LSQAGIDDYNHIIGGHTTEDGLIQAFNVVLNLRHQKDPGFEKIQFK 15139718 QLYKQILSVRTSKSYIPKQFDNSKEMVDCICDYVSKIEKSETVERAL KLVRNISSFDLRGIFVNKKNLRILSNKLIGDWDAIETALMHSSSSEN DKKSVYDSAEAFTLDDIFSSVKKFSDASAEDIGNRAEDICRVISETA PFINDLRAVDLDSLNDDGYEAAVSKIRESLEPYMDLFHELEIFSVG DEFPKCAAFYSELEEVSEQLIEIIPLFNKARSFCTRKRYSTDKIKVNL KFPTLADGWDLNKERDNKAAILRKDGKYYLAILDMKKDLSSIRTS DEDESSFEKMEYKLLPSPVKMLPKIFVKSKAAKEKYGLTDRMLEC YDKGMHKSGSAFDLGFCHELIDYYKRCIAEYPGWDVFDFKFRETS DYGSMKEFNEDVAGAGYYMSLRKIPCSEVYRLLDEKSIYLFQIYN KDYSENAHGNKNMHTMYWEGLFSPQNLESPVFKLSGGAELFFRK SSIPNDAKTVHPKGSVLVPRNDVNGRRIPDSIYRELTRYFNRGDCRI SDEAKSYLDKVKTKKADHDIVKDRRFTVDKMMFHVPIAMNFKAI SKPNLNKKVIDGIIDDQDLKIIGIDRGERNLIYVTMVDRKGNILYQD SLNILNGYDYRKALDVREYDNKEARRNWTKVEGIRKMKEGYLSL AVSKLADMIIENNAIIVMEDLNHGFKAGRSKIEKQVYQKFESMLIN KLGYMVLKDKSIDQSGGALHGYQLANHVTTLASVGKQCGVIFYIP AAFTSKIDPTTGFADLFALSNVKNVASMREFFSKMKSVIYDKAEG KFAFTFDYLDYNVKSECGRTLWTVYTVGERFTYSRVNREYVRKV PTDIIYDALQKAGISVEGDLRDRIAESDGDTLKSIFYAFKYALDMR VENREEDYIQSPVKNASGEFFCSKNAGKSLPQDSDANGAYNIALK GILQLRMLSEQYDPNAESIRLPLITNKAWLTFMQSGMKTWKN

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with LbCas12a): K538A, K538D, K538E, Y542A, Y542D, Y542E, or K595A, K595D, K595E relative to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with AsCas12a): K548A, K548D, K548E, N552A, N552D, N552E, or K607A, K607D, K607 relative to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with CtCas12a): K534A, K534D, K534E, Y538A, Y538D, Y538E, or R591A, R591D, R591E relative to the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with EeCas12a): K542A, K541D, K541E, N545A, N545D, N545E or K601A, K601D, K601E relative to the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with Mb3Cas12a): K579A, K579D, K579E, N583A, N583D, N583E or K635A, K635D, K635E relative to the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with FnCas12a): K613A, K613D, K613E, N617A, N617D, N617E or K671A, K671D, K671E relative to the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with FnoCas12a): K613A, K613D, K613E, N617A, N617D, N617E or N671A, N671D, N671E relative to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with FbCas12a): K617A, K617D, K617E, N621A, N621D, N621E or K678A, K678D, K678E relative to the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with Lb4Cas12a): K541A, K541D, K541E, N545A, N545D, N545E or K601A, K601D, K601E relative to the amino acid sequence of SEQ ID NO: 9.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with MbCas12a): K569A, K569D, K569E, N573A, N573D, N573E or K625A, K625D, K625E relative to the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with Pb2Cas12a): K562A, K562D, K562E, N566A, N566D, N566E or K619A, K619D, K619E relative to the amino acid sequence of SEQ ID NO: 11.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with PgCas12a): K645A, K645D, K645E, N649A, N649D, N649E or K732A, K732D, K732E relative to the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with AaCas12a): K548A, K548D, K548E, N552A, N552D, N552E or K607A, K607D, K607E relative to the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with BoCas12a): K592A, K592D, K592E, N596A, N596D, N596E or K653A, K653D, K653E relative to the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with CMaCas12a): K521A, K521D, K521E, K525A, K525D, K525E or K577A, K577D, K577E relative to the amino acid sequence of SEQ ID NO: 15.

The mutations described herein may be described in the context of a natural Cas12a (any one of SEQ ID NOs: 15) sequence and mutational positions can be carried out by aligning the amino acid sequence of a Cas12a nucleic acid-guided nuclease with, for example, SEQ ID NO: 1 and making the equivalent modification (e.g., substitution) at the equivalent position. By way of example, Table 8 illustrates the equivalent amino acid positions of fifteen orthologous Cas12a nucleic acid-guided nucleases (SEQ ID NOs: 1-15). Any one of the amino acids indicated in Table 8 may be mutated (i.e., via a comparable amino acid substitution).

TABLE 8 Equivalent amino acid positions in homologous Cas12a nucleic acid-guided nuclease Cas 12a AA AA AA AA WT SEQ ID NO Ortholog position position position position SEQ ID NO: 1 LbCas12a G532 K538 Y542 K595 SEQ ID NO: 2 AsCas12a S542 K548 N552 K607 SEQ ID NO: 3 CtCas12a N528 K534 Y538 R591 SEQ ID NO: 4 EeCas12a N535 K541 N545 K601 SEQ ID NO: 5 Mb3Cas12a N573 K579 N583 K635 SEQ ID NO: 6 FnCas12a N607 K613 N617 K671 SEQ ID NO: 7 FnoCas12a N607 K613 N617 N671 SEQ ID NO: 8 FbCas12a N611 K617 N621 K678 SEQ ID NO: 9 Lb4Cas12a N535 K541 N545 K601 SEQ ID NO: 10 MbCas12a N563 K569 N573 K625 SEQ ID NO: 11 Pb2Cas12a G556 K562 N566 K619 SEQ ID NO: 12 PgCas12a D639 K645 N649 K732 SEQ ID NO: 13 AaCas12a S542 K548 N552 K607 SEQ ID NO: 14 BoCas12a K586 K592 N596 K653 SEQ ID NO: 15 CMaCas12a D515 K521 N525 K577

The variant single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-15 (excluding the residues listed in Table 8) and contain any conservative mutation one or more residues indicated in Tables 9-13.

It should be appreciated that any of the amino acid mutations described herein, (e.g., K595A) from a first amino acid residue (e.g., K, an amino acid with a basic side chain) to a second amino acid residue (e.g., A, an amino acid with an aliphatic side chain) may also include mutations from the first amino acid residue, lysine, to an amino acid residue that is similar to (e.g., conserved) the second amino acid residue, alanine, such as valine or glycine. As another example, mutation of an amino acid with a positively charged side chain (e.g., arginine, histidine, or lysine) may be a mutation to a second amino acid with an acidic side chain (e.g., glutamic acid or aspartic acid). As another example, mutation of an amino acid with a polar side chain (e.g., serine, threonine, asparagine, or glutamine) may be a mutation to a second amino acid with a positively charged side chain (e.g., arginine, histidine, or lysine). The skilled artisan would recognize that such conservative amino acid substitutions will likely have minor effects on protein structure and are likely to be well tolerated without compromising function. That is, a mutation from one amino acid to a threonine may be an amino acid mutation to a serine; a mutation from one amino acid to an arginine may be an amino acid mutation to a lysine; a mutation from one amino acid to an isoleucine, may be an amino acid mutation to an alanine, valine, methionine, or leucine; a mutation from one amino acid to a lysine may be an amino acid mutation to an arginine; a mutation from one amino acid to an aspartic acid may be an amino acid mutation to a glutamic acid or asparagine; a mutation from one amino acid to a valine may be an amino acid mutation to an alanine, isoleucine, methionine, or leucine; a mutation from one amino acid to a glycine may be an amino acid mutation to an alanine. It should be appreciated, however, that additional conserved amino acid residues would be recognized by the skilled artisan and any of the amino acid mutations to other conserved amino acid residues are also within the scope of this disclosure.

Exemplary variant Cas12a orthologs are shown in tables 9-13.

TABLE 9 Exemplary Variant Ortholog Cas12a's Variant LbCas12a Variant AsCas12a Variant CtCas12a SEQ (in relation to wt SEQ (in relation to wt SEQ (in relation to wt ID LbCas12a SEQ ID ID AsCas12a SEQ ID ID CtCas12a SEQ ID NO: NO:1) NO: NO:2) NO: NO:3) 16 K595A 55 K607A  94 R591A 17 K595D 56 K607D  95 R591D 18 K595E 57 K607E  96 R591E 19 K538A/K595A 58 K548A/K607A  97 K534A/R591A 20 K538A/K595D 59 K548A/K607D  98 K534A/R591D 21 K538A/K595E 60 K548A/K607E  99 K534A/R591E 22 K538D/K595A 61 K548D/K607A 100 K534D/R591A 23 K538D/K595D 62 K548D/K607D 101 K534D/R591D 24 K538D/K595E 63 K548D/K607E 102 K534D/R591E 25 K538E/K595A 64 K548E/K607A 103 K534E/R591A 26 K538E/K595D 65 K548E/K607D 104 K534E/R591D 27 K538E/K595E 66 K548E/K607E 105 K534E/R591E 28 K538A/Y542A/K595A 67 K548A/N552A/K607A 106 K534A/Y538A/R591A 29 K538A/Y542D/K595A 68 K548A/N552D/K607A 107 K534A/Y538D/R591A 30 K538A/Y542E/K595A 69 K548A/N552E/K607A 108 K534A/Y538E/R591A 31 K538A/Y542A/K595D 70 K548A/N552A/K607D 109 K534A/Y538A/R591D 32 K538A/Y542D/K595D 71 K548A/N552D/K607D 110 K534A/Y538D/R591D 33 K538A/Y542E/K595D 72 K548A/N552E/K607D 111 K534A/Y538E/R591D 34 K538A/Y542A/K595E 73 K548A/N552A/K607E 112 K534A/Y538A/R591E 35 K538A/Y542D/K595E 74 K548A/N552D/K607E 113 K534A/Y538D/R591E 36 K538A/Y542E/K595E 75 K548A/N552E/K607E 114 K534A/Y538E/R591E 37 K538D/Y542A/K595A 76 K548D/N552A/K607A 115 K534D/Y538A/R591A 38 K538D/Y542D/K595A 77 K548D/N552D/K607A 116 K534D/Y538D/R591A 39 K538D/Y542E/K595A 78 K548D/N552E/K607A 117 K534D/Y538E/R591A 40 K538D/Y542A/K595D 79 K548D/N552A/K607D 118 K534D/Y538A/R591D 41 K538D/Y542D/K595D 80 K548D/N552D/K607D 119 K534D/Y538D/R591D 42 K538D/Y542E/K595D 81 K548D/N552E/K607D 120 K534D/Y538E/R591D 43 K538D/Y542A/K595E 82 K548D/N552A/K607E 121 K534D/Y538A/R591E 44 K538D/Y542D/K595E 83 K548D/N552D/K607E 122 K534D/Y538D/R591E 45 K538D/Y542E/K595E 84 K548D/N552E/K607E 123 K534D/Y538E/R591E 46 K538E/Y542A/K595A 85 K548E/N552A/K607A 124 K534E/Y538A/R591A 47 K538E/Y542D/K595A 86 K548E/N552D/K607A 125 K534E/Y538D/R591A 48 K538E/Y542E/K595A 87 K548E/N552E/K607A 126 K534E/Y538E/R591A 49 K538E/Y542A/K595E 88 K548E/N552A/K607D 127 K534E/Y538A/R591D 50 K538E/Y542D/K595E 89 K548E/N552D/K607D 128 K534E/Y538D/R591D 51 K538E/Y542E/K595E 90 K548E/N552E/K607D 129 K534E/Y538E/R591D 52 K538E/Y542A/K595E 91 K548E/N552A/K607E 130 K534E/Y538A/R591E 53 K538E/Y542D/K595E 92 K548E/N552D/K607E 131 K534E/Y538D/R591E 54 K538E/Y542E/K595E 93 K548E/N552E/K607E 132 K534E/Y538E/R591E

TABLE 10 Exemplary Variant Ortholog Cas12a's Variant EeCas12a Variant Mb3Cas12a Variant FnCas12a SEQ (in relation to wt SEQ (in relation to wt SEQ (in relation to wt ID EeCas12a SEQ ID ID Mb3Cas12a SEQ ID ID FnCas12a SEQ ID NO: NO:4) NO: NO:5) NO: NO:6) 133 K601A 172 K635A 211 K671A 134 K601D 173 K635D 212 K671D 135 K601E 174 K635E 213 K671E 136 K541A/K601A 175 K579A/K635A 214 K613A/K671A 137 K541A/K601D 176 K579A/K635D 215 K613A/K671D 138 K541A/K601E 177 K579A/K635E 216 K613A/K671E 139 K541D/K601A 178 K579D/K635A 217 K613D/K671A 140 K541D/K601D 179 K579D/K635D 218 K613D/K671D 141 K541D/K601E 180 K579D/K635E 219 K613D/K671E 142 K541E/K601A 181 K579E/K635A 220 K613E/K671A 143 K541E/K601D 182 K579E/K635D 221 K613E/K671D 144 K541E/K601E 183 K579E/K635E 222 K613E/K671E 145 K541A/N545A/K601A 184 K579A/N583A/K635A 223 K613A/N617A/K671A 146 K541A/N545D/K601A 185 K579A/N583D/K635A 224 K613A/N617D/K671A 147 K541A/N545E/K601A 186 K579A/N583E/K635A 225 K613A/N617E/K671A 148 K541A/N545A/K601D 187 K579A/N583A/K635D 226 K613A/N617A/K671D 149 K541A/N545D/K601D 188 K579A/N583D/K635D 227 K613A/N617D/K671D 150 K541A/N545E/K601D 189 K579A/N583E/K635D 228 K613A/N617E/K671D 151 K541A/N545A/K601E 190 K579A/N583A/K635E 229 K613A/N617A/K671E 152 K541A/N545D/K601E 191 K579A/N583D/K635E 230 K613A/N617D/K671E 153 K541A/N545E/K601E 192 K579A/N583E/K635E 231 K613A/N617E/K671E 154 K541D/N545A/K601A 193 K579D/N583A/K635A 232 K613D/N617A/K671A 155 K541D/N545D/K601A 194 K579D/N583D/K635A 233 K613D/N617D/K671A 156 K541D/N545E/K601A 195 K579D/N583E/K635A 234 K613D/N617E/K671A 157 K541D/N545A/K601D 196 K579D/N583A/K635D 235 K613D/N617A/K671D 158 K541D/N545D/K601D 197 K579D/N583D/K635D 236 K613D/N617D/K671D 159 K541D/N545E/K601D 198 K579D/N583E/K635D 237 K613D/N617E/K671D 160 K541D/N545A/K601E 199 K579D/N583A/K635E 238 K613D/N617A/K671E 161 K541D/N545D/K601E 200 K579D/N583D/K635E 239 K613D/N617D/K671E 162 K541D/N545E/K601E 201 K579D/N583E/K635E 240 K613D/N617E/K671E 163 K541E/N545A/K601A 202 K579E/N583A/K635A 241 K613E/N617A/K671A 164 K541E/N545D/K601A 203 K579E/N583D/K635A 242 K613E/N617D/K671A 165 K541E/N545E/K601A 204 K579E/N583E/K635A 243 K613E/N617E/K671A 166 K541E/N545A/K601D 205 K579E/N583A/K635D 244 K613E/N617A/K671D 167 K541E/N545D/K601D 206 K579E/N583D/K635D 245 K613E/N617D/K671D 168 K541E/N545E/K601D 207 K579E/N583E/K635D 246 K613E/N617E/K671D 169 K541E/N545A/K601E 208 K579E/N583A/K635E 247 K613E/N617A/K671E 170 K541E/N545D/K601E 209 K579E/N583D/K635E 248 K613E/N617D/K671E 171 K541E/N545E/K601E 210 K579E/N583E/K635E 249 K613E/N617E/K671E

TABLE 11 Exemplary Variant Ortholog Cas12a's Variant FnoCas12a Variant FbCas12a Variant Lb4as12a SEQ (in relation to wt SEQ (in relation to wt SEQ (in relation to wt ID FnoCas12a SEQ ID ID FbCas12a SEQ ID ID Lb4Cas12a SEQ ID NO: NO:7) NO: NO:8) NO: NO:9) 250 N671A 289 K678A 328 K601A 251 N671D 290 K678D 329 K601D 252 N671E 291 K678E 330 K601E 253 K613A/N671A 292 K617A/K678A 331 K541A/K601A 254 K613A/N671D 293 K617A/K678D 332 K541A/K601D 255 K613A/N671E 294 K617A/K678E 333 K541A/K601E 256 K613D/N671A 295 K617D/K678A 334 K541D/K601A 257 K613D/N671D 296 K617D/K678D 335 K541D/K601D 258 K613D/N671E 297 K617D/K678E 336 K541D/K601E 259 K613E/N671A 298 K617E/K678A 337 K541E/K601A 260 K613E/N671D 299 K617E/K678D 338 K541E/K601D 261 K613E/N671E 300 K617E/K678E 339 K541E/K601E 262 K613A/N617A/N671A 301 K617A/N621A/K678A 340 K541A/N545A/K601A 263 K613A/N617D/N671A 302 K617A/N621D/K678A 341 K541A/N545D/K601A 264 K613A/N617E/N671A 303 K617A/N621E/K678A 342 K541A/N545E/K601A 265 K613A/N617A/N671D 304 K617A/N621A/K678D 343 K541A/N545A/K601D 266 K613A/N617D/N671D 305 K617A/N621D/K678D 344 K541A/N545D/K601D 267 K613A/N617E/N671D 306 K617A/N621E/K678D 345 K541A/N545E/K601D 268 K613A/N617A/N671E 307 K617A/N621A/K678E 346 K541A/N545A/K601E 269 K613A/N617D/N671E 308 K617A/N621D/K678E 347 K541A/N545D/K601E 270 K613A/N617E/N671E 309 K617A/N621E/K678E 348 K541A/N545E/K601E 271 K613D/N617A/N671A 310 K617D/N621A/K678A 349 K541D/N545A/K601A 272 K613D/N617D/N671A 311 K617D/N621D/K678A 350 K541D/N545D/K601A 273 K613D/N617E/N671A 312 K617D/N621E/K678A 351 K541D/N545E/K601A 274 K613D/N617A/N671D 313 K617D/N621A/K678D 352 K541D/N545A/K601D 275 K613D/N617D/N671D 314 K617D/N621D/K678D 353 K541D/N545D/K601D 276 K613D/N617E/N671D 315 K617D/N621E/K678D 354 K541D/N545E/K601D 277 K613D/N617A/N671E 316 K617D/N621A/K678E 355 K541D/N545A/K601E 278 K613D/N617D/N671E 317 K617D/N621D/K678E 356 K541D/N545D/K601E 279 K613D/N617E/N671E 318 K617D/N621E/K678E 357 K541D/N545E/K601E 280 K613E/N617A/N671A 319 K617E/N621A/K678A 358 K541E/N545A/K601A 281 K613E/N617D/N671A 320 K617E/N621D/K678A 359 K541E/N545D/K601A 282 K613E/N617E/N671A 321 K617E/N621E/K678A 360 K541E/N545E/K601A 283 K613E/N617A/N671D 322 K617E/N621A/K678D 361 K541E/N545A/K601D 284 K613E/N617D/N671D 323 K617E/N621D/K678D 362 K541E/N545D/K601D 285 K613E/N617E/N671D 324 K617E/N621E/K678D 363 K541E/N545E/K601D 286 K613E/N617A/N671E 325 K617E/N621A/K678E 364 K541E/N545A/K601E 287 K613E/N617D/N671E 326 K617E/N621D/K678E 365 K541E/N545D/K601E 288 K613E/N617E/N671E 327 K617E/N621E/K678E 366 K541E/N545E/K601E

TABLE 12 Exemplary Variant Ortholog Cas12a's Variant MbCas12a Variant Pb2Cas12a Variant PgCas12a SEQ (in relation to wt SEQ (in relation to wt SEQ (in relation to wt ID MbCas12a SEQ ID ID Pb2Cas12a SEQ ID ID PgCas12a SEQ ID NO: NO:10) NO: NO:11) NO: NO:12) 367 K625A 406 K619A 445 K732A 368 K625D 407 K619D 446 K732D 369 K625E 408 K619E 447 K732E 370 K569A/K625A 409 K562A/K619A 448 K645A/K732A 371 K569A/K625D 410 K562A/K619D 449 K645A/K732D 372 K569A/K625E 411 K562A/K619E 450 K645A/K732E 373 K569D/K625A 412 K562D/K619A 451 K645D/K732A 374 K569D/K625D 413 K562D/K619D 452 K645D/K732D 375 K569D/K625E 414 K562D/K619E 453 K645D/K732E 376 K569E/K625A 415 K562E/K619A 454 K645E/K732A 377 K569E/K625D 416 K562E/K619D 455 K645E/K732D 378 K569E/K625E 417 K562E/K619E 456 K645E/K732E 379 K569A/N573A/K625A 418 K562A/N566A/K619A 457 K645A/N649A/K732A 380 K569A/N573D/K625A 419 K562A/N566D/K619A 458 K645A/N649D/K732A 381 K569A/N573E/K625A 420 K562A/N566E/K619A 459 K645A/N649E/K732A 382 K569A/N573A/K625D 421 K562A/N566A/K619D 460 K645A/N649A/K732D 383 K569A/N573D/K625D 422 K562A/N566D/K619D 461 K645A/N649D/K732D 384 K569A/N573E/K625D 423 K562A/N566E/K619D 462 K645A/N649E/K732D 385 K569A/N573A/K625E 424 K562A/N566A/K619E 463 K645A/N649A/K732E 386 K569A/N573D/K625E 425 K562A/N566D/K619E 464 K645A/N649D/K732E 387 K569A/N573E/K625E 426 K562A/N566E/K619E 465 K645A/N649E/K732E 388 K569D/N573A/K625A 427 K562D/N566A/K619A 466 K645D/N649A/K732A 389 K569D/N573D/K625A 428 K562D/N566D/K619A 467 K645D/N649D/K732A 390 K569D/N573E/K625A 429 K562D/N566E/K619A 468 K645D/N649E/K732A 391 K569D/N573A/K625D 430 K562D/N566A/K619D 469 K645D/N649A/K732D 392 K569D/N573D/K625D 431 K562D/N566D/K619D 470 K645D/N649D/K732D 393 K569D/N573E/K625D 432 K562D/N566E/K619D 471 K645D/N649E/K732D 394 K569D/N573A/K625E 433 K562D/N566A/K619E 472 K645D/N649A/K732E 395 K569D/N573D/K625E 434 K562D/N566D/K619E 473 K645D/N649D/K732E 396 K569D/N573E/K625E 435 K562D/N566E/K619E 474 K645D/N649E/K732E 397 K569E/N573A/K625A 436 K562E/N566A/K619A 475 K645E/N649A/K732A 398 K569E/N573D/K625A 437 K562E/N566D/K619A 476 K645E/N649D/K732A 399 K569E/N573E/K625A 438 K562E/N566E/K619A 477 K645E/N649E/K732A 400 K569E/N573A/K625D 439 K562E/N566A/K619D 478 K645E/N649A/K732D 401 K569E/N573D/K625D 440 K562E/N566D/K619D 479 K645E/N649D/K732D 402 K569E/N573E/K625D 441 K562E/N566E/K619D 480 K645E/N649E/K732D 403 K569E/N573A/K625E 442 K562E/N566A/K619E 481 K645E/N649A/K732E 404 K569E/N573D/K625E 443 K562E/N566D/K619E 482 K645E/N649D/K732E 405 K569E/N573E/K625E 444 K562E/N566E/K619E 483 K645E/N649E/K732E

TABLE 13 Exemplary Variant Ortholog Cas12a's Variant AaCas12a Variant BoCas12a Variant CMaCas12a SEQ (in relation to wt SEQ (in relation to wt SEQ (in relation to wt ID AaCas12a SEQ ID ID BoCas12a SEQ ID ID CMaCas12a SEQ ID NO: NO:13) NO: NO:14) NO: NO:15) 484 K607A 523 K653A 562 K577A 485 K607D 524 K653D 563 K577D 486 K607E 525 K653E 564 K577E 487 K548A/K607A 526 K592A/K653A 565 K521A/K577A 488 K548A/K607D 527 K592A/K653D 566 K521A/K577D 489 K548A/K607E 528 K592A/K653E 567 K521A/K577E 490 K548D/K607A 529 K592D/K653A 568 K521D/K577A 491 K548D/K607D 530 K592D/K653D 569 K521D/K577D 492 K548D/K607E 531 K592D/K653E 570 K521D/K577E 493 K548E/K607A 532 K592E/K653A 571 K521E/K577A 494 K548E/K607D 533 K592E/K653D 572 K521E/K577D 495 K548E/K607E 534 K592E/K653E 573 K521E/K577E 496 K548A/N552A/K607A 535 K592A/N596A/K653A 574 K521A/N525A/K577A 497 K548A/N552D/K607A 536 K592A/N596D/K653A 575 K521A/N525D/K577A 498 K548A/N552E/K607A 537 K592A/N596E/K653A 576 K521A/N525E/K577A 499 K548A/N552A/K607D 538 K592A/N596A/K653D 577 K521A/N525A/K577D 500 K548A/N552D/K607D 539 K592A/N596D/K653D 578 K521A/N525D/K577D 501 K548A/N552E/K607D 540 K592A/N596E/K653D 579 K521A/N525E/K577D 502 K548A/N552A/K607E 541 K592A/N596A/K653E 580 K521A/N525A/K577E 503 K548A/N552D/K607E 542 K592A/N596D/K653E 581 K521A/N525D/K577E 504 K548A/N552E/K607E 543 K592A/N596E/K653E 582 K521A/N525E/K577E 505 K548D/N552A/K607A 544 K592D/N596A/K653A 583 K521D/N525A/K577A 506 K548D/N552D/K607A 545 K592D/N596D/K653A 584 K521D/N525D/K577A 507 K548D/N552E/K607A 546 K592D/N596E/K653A 585 K521D/N525E/K577A 508 K548D/N552A/K607D 547 K592D/N596A/K653D 586 K521D/N525A/K577D 509 K548D/N552D/K607D 548 K592D/N596D/K653D 587 K521D/N525D/K577D 510 K548D/N552E/K607D 549 K592D/N596E/K653D 588 K521D/N525E/K577D 511 K548D/N552A/K607E 550 K592D/N596A/K653E 589 K521D/N525A/K577E 512 K548D/N552D/K607E 551 K592D/N596D/K653E 590 K521D/N525D/K577E 513 K548D/N552E/K607E 552 K592D/N596E/K653E 591 K521D/N525E/K577E 514 K548E/N552A/K607A 553 K592E/N596A/K653A 592 K521E/N525A/K577A 515 K548E/N552D/K607A 554 K592E/N596D/K653A 593 K521E/N525D/K577A 516 K548E/N552E/K607A 555 K592E/N596E/K653A 594 K521E/N525E/K577A 517 K548E/N552A/K607D 556 K592E/N596A/K653D 595 K521E/N525A/K577D 518 K548E/N552D/K607D 557 K592E/N596D/K653D 596 K521E/N525D/K577D 519 K548E/N552E/K607D 558 K592E/N596E/K653D 597 K521E/N525E/K577D 520 K548E/N552A/K607E 559 K592E/N596A/K653E 598 K521E/N525A/K577E 521 K548E/N552D/K607E 560 K592E/N596D/K653E 599 K521E/N525D/K577E 522 K548E/N552E/K607E 561 K592E/N596E/K653E 600 K521E/N525E/K577E

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 70% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 75% identical to any one of SEQ ID NOs: 16-600 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 80% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 85% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 90% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 95% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is any one of SEQ ID NOs: 16-600.

The mutations described herein are described in the context of the WT LbCas12a (e.g., SEQ ID NO: 1) sequence and mutational positions can be carried out by aligning the amino acid sequence of a Cas12a nucleic acid-guided nuclease with SEQ ID NO: 1 and making the equivalent modification (e.g., substitution) at the equivalent position. By way of example, the mutations described herein may be applied to a Cas12a enzyme shown in Table 7, or any other homolog Cas12a thereof by aligning the amino acid sequence of the Cas12a to SEQ ID NO: 1 and making the modifications described in Tables 9-13 (changes to the wildtype residue to alanine, aspartic acid or glutamic acid or conservative equivalents at the Cas12a ortholog's equivalent position (e.g., see Table 8 for an example of equivalent residue positions).

For example, in addition to the variant LbCas12a sequences in Table 9 (variant sequences SEQ ID Nos: 16-54), like variants are envisioned for AsCas12a (variant sequences SEQ ID Nos: 55-93), CtCas12a (variant sequences SEQ ID Nos: 94-132), EeCas12a (variant sequences SEQ ID Nos: 133-171), Mb3Cas12a (variant sequences SEQ ID Nos: 172-210), FnCas12a (variant sequences SEQ ID Nos: 211-249), FnoCas12a (variant sequences SEQ ID Nos: 250-288), FbCas12a (variant sequences SEQ ID Nos: 289-327), Lb4Cas12a (variant sequences SEQ ID Nos: 328-366), MbCas12a (variant sequences SEQ ID Nos: 367-405), Pb2Cas12a (variant sequences SEQ ID Nos: 406-444), PgCas12a (variant sequences SEQ ID Nos: 445-483), AaCas12a (variant sequences SEQ ID Nos: 484-522), BoCas12a (variant sequences SEQ ID Nos: 523-561), and CmaCas12a (variant sequences SEQ ID Nos: 562-600). In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 70% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 75% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 80% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 85% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 90% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 95% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least %, 97%, 98% or 99% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is any one of SEQ ID NOs: 16-600.

The single-strand-specific Cas12a nucleic acid-guided nucleases described herein may be any Cas12a nucleic acid-guided nuclease that largely prevents double-stranded nucleic acid unwinding and R-loop formation. The single-strand-specific Cas12a nucleic acid-guided nucleases described herein may also be any Cas12a nucleic acid-guided nuclease that lacks cis-cleavage activity yet maintains trans-nucleic acid-guided nuclease activity on single-stranded nucleic acid molecules. Such single-strand-specific Cas12a nucleic acid-guided nucleases may be generated via the mutations described herein.

Additionally, or alternatively, such single-strand-specific Cas12a nucleic acid-guided nucleases may be generated via post-translational modifications (e.g., acetylation). The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an acetylated Cas12a enzyme. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an LbCas12a (i.e., SEQ ID NO: 1) with an acetylated K595 (K595K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an AsCas12a (i.e., SEQ ID NO: 2) with an acetylated K607 (K607K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a CtCas12a (i.e., SEQ ID NO: 3) with an acetylated R591 (R591R^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an EeCas12a (i.e., SEQ ID NO: 4) with an acetylated K601 (K607K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an Mb3Cas12a (i.e., SEQ ID NO: 5) with an acetylated K635 (K635K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an FnCas12a (i.e., SEQ ID NO: 6) with an acetylated K671 (K671K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an FnoCas12a (i.e., SEQ ID NO: 7) with an acetylated N671 (N671K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an FbCas12a (i.e., SEQ ID NO: 8) with an acetylated K678 (K678K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an Lb4Cas12a (i.e., SEQ ID NO: 9) with an acetylated K601 (K601K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an MbCas12a (i.e., SEQ ID NO: 10) with an acetylated K625 (K625K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a Pb2Cas12a (i.e., SEQ ID NO: 11) with an acetylated K619 (K619K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a PgCas12a (i.e., SEQ ID NO: 12) with an acetylated K732 (K732K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an AaCas12a (i.e., SEQ ID NO: 13) with an acetylated K607 (K607K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an BoCas12a (i.e., SEQ ID NO: 14) with an acetylated K653 (K653K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an CmaCas12a (i.e., SEQ ID NO: 15) with an acetylated K577 (K577K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a Cas12a ortholog acetylated at the amino acid of the ortholog equivalent to K595 of SEQ ID NO:1. Acetylation of Cas12a can be carried out with any suitable acetyltransferase. For a discussion and methods for disabling of Cas12a by ArVA5, see Dong, et al., Nature Structural and Molecular Bio., 26(4):308-14 (2019). For example, LbCas12a can be incubated with AcrVA5 in order to acetylate the K595 residue, thereby deactivating the dsDNA activity (e.g., FIG. 7 ). In addition to acetylation, phosphorylation and methylation of select amino acid residues may be employed.

Bulky Modifications

In addition to the modalities of adjusting the ratio of the concentration of the blocked nucleic acid molecules to the concentration of the RNP2 and altering the domains of the variant nucleic acid-guided nuclease of RNP2 that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules to vary dsDNA vs. ssDNA recognition properties as described in detail above, the present disclosure additionally contemplates use of “bulky modifications” at the 5′ and/or 3′ ends and/or at internal nucleic acid bases of the blocked nucleic acid molecule and/or using modifications between internal nucleic acid bases. FIG. 8A is an illustration of the steric hindrance at the PAM-interacting (PI) domain in a nucleic acid-guided nuclease caused by 5′ and 3′ modifications to a blocked nucleic acid molecule. At top in FIG. 8A is an illustration of the target stand and non-target strand, and below this is an illustration of a self-hybridized blocked nucleic acid molecule comprising three loop regions, as well as bulky modifications on the 5′ and 3′ ends of the blocked nucleic acid molecule. Example “bulky modifications” include a fluorophore and quencher pair (as shown here) or biotin, but in general encompass molecules with a size of about 1 nm or less, or 0.9 nm or less, or 0.8 nm or less, or 0.7 nm or less, or 0.6 nm or less, or 0.5 nm or less, or 0.4 nm or less, or 0.3 nm or less, or 0.2 nm or less, or 0.1 nm or less, or 0.05 nm or less, or as small as 0.025 nm or less.

In the illustration at center, the blocked nucleic acid molecule with the 5′ and 3′ ends comprising a fluorophore and a quencher is shown being cleaved at the loop regions. Note that the bulky modifications in this embodiment also allow the blocked nucleic acid molecule to act as a reporter moiety; that is, when the loop regions of the blocked nucleic acid molecule are cleaved, the short nucleotide segments of the non-target strand dehybridize from the target strand due to low T_(m), thereby separating the fluorophore and quencher such that fluorescence from the fluorophore is no longer quenched and can be detected. In the illustration at bottom, the intact blocked nucleic acid molecule with the bulky modifications (at left) sterically hinders interaction with the PAM-interacting (PI) domain of the nucleic acid-guided nuclease in RNP2 such that the intact blocked nucleic acid molecule cannot be cleaved via cis-cleavage by the nucleic acid-guided nuclease. However, once the loop regions of the blocked nucleic acid molecule are cleaved (via, e.g., trans-cleavage from RNP1 (at right)) and the short nucleotide segments of the non-target strand dehybridize from the target strand, leaving the 3′ end of the now single-stranded target strand is now free to initiate R-loop formation with RNP2. R-loop formation leads to cis-cleavage of the single-strand target strand, and subsequent activation of trans-cleavage of RNP2.

FIG. 8B illustrates five exemplary variations of blocked nucleic acid molecules with bulky modifications, including at the 5′ and/or 3′ ends of a self-hybridizing blocked nucleic acid molecule and/or at internal nucleic acid bases of the blocked nucleic acid molecule. Embodiment (i) illustrates a self-hybridizing blocked nucleic acid molecule having a fluorophore at its 5′ end and a quencher at its 3′ end. Embodiment (ii) illustrates a self-hybridizing blocked nucleic acid molecule having a fluorophore and a quencher at internal nucleic acid bases flanking a loop sequence. Embodiment (iii) illustrates a self-hybridizing blocked nucleic acid molecule having a fluorophore at its 5′ end and a quencher at its 3′ end as well as having a fluorophore and a quencher at internal nucleic acid bases where the internal fluorophore and quencher flank a loop sequence. The fluorophore/quencher embodiments work as long as the fluorophore and quencher are at a distance of about 10-11 nm or less apart. Embodiment (iv) illustrates a self-hybridizing blocked nucleic acid molecule having a biotin molecule at its 5′ end, and embodiment (v) illustrates a self-hybridizing blocked nucleic acid molecule having a biotin at an internal nucleic acid base. Note that bulky modifications of internal nucleic acid bases often are made at or near a loop region of a blocked nucleic acid molecule (or blocked target molecule). The loop regions are regions of the blocked nucleic acid molecules—in addition to the 5′ and 3′ ends—that may be vulnerable to unwinding.

Modifications can be used in self-hybridized blocked nucleic acid molecules lacking a PAM or those comprising a PAM, partially self-hybridized blocked nucleic acid molecules lacking a PAM or those comprising a PAM, or reverse PAM molecules. Other variations include using RNA loops instead of DNA loops if a Cas 13 nucleic acid-guided nuclease is used as the nucleic acid-guided nuclease in RNP1, or entire RNA molecules if a Cas 13 nucleic acid-guided nuclease is used as the nucleic acid-guided nuclease in RNP1 and RNP2.

FIGS. 8C, 8D and 8E list exemplary bulky modifications for 5′, 3′, and internal positions in blocked nucleic acid molecules, and Table 14 below lists sequences of exemplary self-hybridizing blocked nucleic acid molecules. 56-FAM stands for 5′ 6-FAM (6-fluorescein amidite); and 3BHQ stands for 3′ BLACK HOLE QUENCHER®-1.

TABLE 14 Bulky Modifications SEQ ID Molecule No. NO: Name Molecule Sequence (5′→3′) 5′ FAM + 3′ BHQ 1 601 5′F_U29_Q /56- FAM/GATCCATTTTATTTTAGATCATATATATACATGATCGG ATC/3BHQ_1/ 2 602 5′F_1C /56- armor_U29_Q FAM/CGATCCATTTTATTTTAGATCATATATATACATGATCG GATCG/3BHQ_1/ 3 603 5′F_2CC /56- armor_U29_Q FAM/CCGATCCATTTTATTTTAGATCATATATATACATGATC GGATCGG/3BHQ_1/ 4 604 5′F_1A /56- armor_U29_Q FAM/AGATCCATTTTATTTTAGATCATATATATACATGATCG GATCT/3BHQ_1/ 5 605 5′F_2AT /56- armor_U29_Q FAM/ATGATCCATTTTATTTTAGATCATATATATACATGATC GGATCAT/3BHQ_1/ 6 606 5′F_U250_Q /56- FAM/GATATATAAAAAAAAAAAGATCATATACATATATGAT CATATATC/3BHQ_1/ 7 607 5′F_1C /56- armor_U250_Q FAM/CGATATATAAAAAAAAAAAGATCATATACATATATGA TCATATATCG/3BHQ_1/ 8 608 5′F_2CC /56- armor_U250_Q FAM/CCGATATATAAAAAAAAAAAGATCATATACATATATG ATCATATATCGG/3BHQ_1/ 9 609 5′F_1A /56- armor_U250_Q FAM/AGATATATAAAAAAAAAAAGATCATATACATATATGA TCATATATCT/3BHQ_1/ 10 610 5′F_2AT /56- armor_U250_Q FAM/ATGATATATAAAAAAAAAAAGATCATATACATATATG ATCATATATCAT/3BHQ_1/ 5′ Fluorsceine (modification on base) + 3′ BHQ 11 611 5′FdT_U29_Q /5FluorT/GATCCATTTTATTTTAGATCATATATATACATGATC GGATCA/3BHQ_1/ 12 612 5′FdT_1C /5FluorT/CGATCCATTTTATTTTAGATCATATATATACATGAT armor_U29_Q CGGATCGA/3BHQ_1/ 13 605 5′FdT_1A A/iFluorT/GATCCATTTTATTTTAGATCATATATATACATGAT armor_U29_Q CGGATCAT/3BHQ_1/ 14 613 5′FdT_U250_Q /5FluorT/GATATATAAAAAAAAAAAGATCATATACATATATG ATCATATATCA/3BHQ_1/ 15 614 5′FdT_1C /5FluorT/CGATATATAAAAAAAAAAAGATCATATACATATAT armor_U250_Q GATCATATATCGA/3BHQ_1/ 16 610 5′FdT_1A A/iFluorT/GATATATAAAAAAAAAAAGATCATATACATATAT armor_U250_Q GATCATATATCAT/3BHQ_1/ 5′ FAM + Internal Fluorsceine (modification on base) + 3′ BHQ 17 601 5′F_IntFdt_U29_Q /56- FAM/GA/iFluorT/CCATTTTATTTTAGATCATATATATACATG ATCGGATC/3BHQ_1/ 18 606 5′F_IntFdt_U250_Q /56- FAM/GA/iFluorT/ATATAAAAAAAAAAAGATCATATACATAT ATGATCATATATC/3BHQ_1/ 19 602 5′F_1C /56- armor_IntFdt_U29_Q FAM/CGA/iFluorT/CCATTTTATTTTAGATCATATATATACAT GATCGGATCG/3BHQ_1/ 20 604 5′F_1A /56- armor_IntFdt_U29_Q FAM/AGA/iFluorT/CCATTTTATTTTAGATCATATATATACAT GATCGGATCT/3BHQ_1/ 21 607 5′F_1C /56- armor_IntFdt_U250_Q FAM/CGA/iFluorT/ATATAAAAAAAAAAAGATCATATACATA TATGATCATATATCG/3BHQ_1/ 22 609 5′F_1A /56- armor_IntFdt_U250_Q FAM/AGA/iFluorT/ATATAAAAAAAAAAAGATCATATACATA TATGATCATATATCT/3BHQ_1/ 23 603 5′F_2CC /56- armor_IntFdt_U29_Q FAM/CCGA/iFluorT/CCATTTTATTTTAGATCATATATATACA TGATCGGATCGG/3BHQ_1/ 24 605 5′F_2AT /56- armor_IntFdt_U29_Q FAM/ATGA/iFluorT/CCATTTTATTTTAGATCATATATATACA TGATCGGATCAT/3BHQ_1/ 25 608 5′F_2CC /56- armor_IntFdt_U250_Q FAM/CCGA/iFluorT/ATATAAAAAAAAAAAGATCATATACAT ATATGATCATATATCGG/3BHQ_1/ 26 610 5′F_2AT /56- armor_IntFdt_U250_Q FAM/ATGA/iFluorT/ATATAAAAAAAAAAAGATCATATACAT ATATGATCATATATCAT/3BHQ_1/ Applications of the Cascade Assay

The present disclosure describes cascade assays for detecting a target nucleic acid of interest in a sample that provide instantaneous or nearly instantaneous results even at ambient temperatures at 16° C. and above, allow for massive multiplexing and minimum workflow, yet provide accurate results at low cost. Moreover, the various embodiments of the cascade assay are notable in that, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected and RNP1 is easily reprogrammed. Moreover, the cascade assay can be massively multiplexed for detecting several to many to target nucleic acid molecules simultaneously. For example, the assay may be designed to detect one to several to many different pathogens (e.g., testing for many different pathogens in one assay), or the assay may be designed to detect one to several to many different sequences from the same pathogen (e.g., to increase specificity and sensitivity), or a combination of the two.

As described above, early and accurate identification of, e.g., infectious agents, microbe contamination, and variant nucleic acid sequences that indicate the present of such diseases such as cancer or contamination by heterologous sources is important in order to select correct therapeutic treatment, identify tainted food, pharmaceuticals, cosmetics and other commercial goods; and to monitor the environment. The cascade assay described herein can be applied in diagnostics for, e.g., infectious disease (including but not limited to Covid, HIV, flu, the common cold, Lyme disease, STDs, chicken pox, diptheria, mononucleosis, hepatitis, UTIs, pneumonia, tetanus, rabies, malaria, dengue fever, Ebola, plague; see Table 1), for rapid liquid biopsies and companion diagnostics (biomarkers for cancers, early detection, progression, monitoring; see Table 4), prenatal testing (including but not limited to chromosomal abnormalities and genetic diseases such as sickle cell, including over-the-counter versions of prenatal testing assays), rare disease testing (achondroplasia, Addison's disease, al-antitrypsin deficiency, multiple sclerosis, muscular dystrophy, cystic fibrosis, blood factor deficiencies), SNP detection/DNA profiling/epigenetics, genotyping, low abundance transcript detection, labeling for cell or droplet sorting, in situ nucleic acid detection, sample prep, library quantification of NGS, screening biologics (including engineered therapeutic cells for genetic integrity and/or contamination), development of agricultural products, food compliance testing and quality control (e.g., detection of genetically modified products, confirmation of source for high value commodities, contamination detection), infectious disease in livestock, infectious disease in cash crops, livestock breeding, drug screening, personal genome testing including clinical trial stratification, personalized medicine, nutrigenomics, drug development and drug therapy efficacy, transplant compatibility and monitoring, environmental testing and forensics, and bioterrorism agent monitoring.

Target nucleic acids of interest are derived from samples as described in more detail above. Suitable samples for testing include, but are not limited to, any environmental sample, such as air, water, soil, surface, food, clinical sites and products, industrial sites and products, pharmaceuticals, medical devices, nutraceuticals, cosmetics, personal care products, agricultural equipment and sites, and commercial samples, and any biological sample obtained from an organism or a part thereof, such as a plant, animal, or microbe. In some embodiments, the biological sample is obtained from an animal subject, such as a human subject. A biological sample may be any solid or fluid sample obtained from, excreted by or secreted by any living organism, including, without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms including plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as an infection with a pathogenic microorganism, such as a pathogenic bacteria or virus.

For example, a biological sample can be a biological fluid obtained from a human or non-human (e.g., livestock, pets, wildlife) animal, and may include but is not limited to blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease, such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or a swab of skin or mucosal membrane surface (e.g., a nasal or buccal swab).

In some embodiments, the sample can be a viral or bacterial sample or a biological sample that has been minimally processed, e.g., only treated with a brief lysis step prior to detection. In other embodiments, minimal processing can include thermal lysis at an elevated temperature to release nucleic acids. Suitable methods are contemplated in U.S. Pat. No. 9,493,736, among other references. Common methods for cell lysis involve thermal, chemical, enzymatic, or mechanical treatment of the sample or a combination of those (see, e.g., Example I below). In some embodiments, minimal processing can include treating the sample with chaotropic salts such as guanidine isothiocyanate or guanidine HCl. Suitable methods are contemplated in U.S. Pat. Nos. 8,809,519 and 7,893,251, among other references. In some embodiments, minimal processing may include contacting the sample with reducing agents such as DTT or TCEP and EDTA to inactivate inhibitors and/or other nucleases present in the crude samples. In other embodiments, minimal processing for biofluids may include centrifuging the samples to obtain cell-debris free supernatant before applying the reagents. Suitable methods are contemplated in U.S. Pat. No. 8,809,519, among other references. In still other embodiments, minimal processing may include performing DNA/RNA extraction to get purified nucleic acids before applying CRISPR Cascade reagents.

Table 15 below lists exemplary commercial sample processing kits, and Table 16 below lists point of care processing techniques.

TABLE 15 Exemplary Commercial Sample and Nucleic Acid Processing Kits Manufacturer Kit Sample Type Output Lysing and extraction methods Qiagen ® DNeasy ™ Blood small volumes genomic Isolation of Genomic DNA from Small & Tissue Kits of blood DNA Volumes of Blood dried blood 1. Uses Chemical and spots Biological/Enzymatic lysis methods urine 2. Uses SPE with Column Purification tissues Isolation of Genomic DNA from Tissues laser- 1. Uses Chemical and microdissected Biological/Enzymatic lysis methods tissues 2. Used to dissolve and lyse tissue sections completely, higher temperature and longer time incubations up to 24 hours are used Qiagen ® QIAamp ® UCP whole blood microbial Specific pretreatment protocols are Pathogen swabs DNA suggested depending on sample type with Mini Handbook cultures -- or without the use of kits for Mechanical microbial DNA pelleted Lysis Method before downstream purification microbial cells applications. body fluids Downstream applications contain: 1. Chemical and Biological/Enzymatic lysis methods 2. SPE with Column Purification Qiagen ® QIAamp ® Viral plasma and viral DNA 1. Uses Chemical lysis methods RNA Kits serum 2. Uses SPE with Column Purification CSF urine other cell-free body fluids cell-culture supernatants swabs Zymo Quick- whole blood genomic 1. Uses chemical lysis methods Research ™ DNA ™ Microprep plasma DNA 2. Uses SPE with column purification Kit serum body fluids buffy coat lymphocytes swabs cultured cells Zymo Quick-DNA ™ A. fumigatus Microbial Uses Bead lysis and pretreatment with: Research ™ Fungal/Bacterial C. albicans DNA 1. Chemical lysis methods with Miniprep Kit N. crassa chaotropic salts S. cerevisiae 2. NAE with SPE with silica matrices S. pombe mycelium Gram positive bacteria Gram negative bacteria

TABLE 16 Point of Care Sample Processing Techniques Steps Protocol Example 1 Protocol Example 2 Protocol Example 3 Field-deployable viral Streamlined Lucira Health ™ diagnostics using inactivation, CRISPR-Cas13 amplification, and Science, Cas13-based detection 27; 360(6387):444-448 of SARS-COV-2 (2018) Nat Commun, 11: 5921 (2020) 1. Cell disruption Samples were thermally A NP swab or saliva Lucira Health uses a (lysis) and treated at ~40° C. for sample was lysed and single buffer that lyses inactivation of ~15 minutes for nuclease inactivated for 10 and inactivates nucleases deactivation, thereafter minutes with thermal nucleases and/or In POC setting, cell at 90° C. for 5 minutes treatment. These inhibitors. disruption and for viral deactivation. samples were incubated A nasal swab is directly inactivation of Sample Types: for 5 min at 40° C., added to a single nucleases is done Urine followed by 5 min at lysing/reaction buffer commonly through Saliva 70° C. (or 5 min at and vigorously stirred thermal lysis. Diluted blood 95° C., if saliva) to release the viral (1:3 with PBS) particulates from the Targets: Viruses swab. Target: SARS-Cov-2 2. Assay on crude Thermally treated Thermally treated Processed biological sample biological biological sample is used in an This is usually a direct samples(above) were samples(above) were isothermal reaction for assay on the crude used directly for used directly for pathogenic nucleic acid sample post cell amplification and amplification and detection. disruption and detection of pathogenic detection of pathogenic inactivation of nucleic acid. nucleic acid. nucleases. No extraction is usually performed.

FIG. 9 shows a lateral flow assay (LFA) device that can be used to detect the cleavage and separation of a signal from a reporter moiety. For example, the reporter moiety may be a single-stranded or double-stranded oligonucleotide with terminal biotin and fluorescein amidite (FAM) modifications; and, as described above, the reporter moiety may also be part of a blocked nucleic acid. The LFA device may include a pad with binding particles, such as gold nanoparticles functionalized with anti-FAM antibodies; a control line with a first binding moiety attached, such as avidin or streptavidin; a test line with a second binding moiety attached, such as antibodies; and an absorption pad. After completion of a cascade assay (see FIGS. 2A, 3A, and 3B), the assay reaction mix is added to the pad containing the binding particles, (e.g., antibody labeled gold nanoparticles). When the target nucleic acid of interest is present, a reporter moiety is cleaved, and when the target nucleic acid of interest is absent, the reporter is not cleaved.

A moiety on the reporter binds to the binding particles and is transported to the control line. When the target nucleic acid of interest is absent, the reporter moiety is not cleaved, and the first binding moiety binds to the reporter moiety, with the binding particles attached. When the target nucleic acid of interest is present, one portion of the cleaved reporter moiety binds to the first binding moiety, and another portion of the cleaved reporter moiety bound to the binding particles via the moiety binds to the second binding moiety. In one example, anti-FAM gold nanoparticles bind to a FAM terminus of a reporter moiety and flow sequentially toward the control line and then to the test line. For reporters that are not trans-cleaved, gold nanoparticles attach to the control line via biotin-streptavidin and result in a dark control line. In a negative test, since the reporter has not been cleaved, all gold conjugates are trapped on control line due to attachment via biotin-streptavidin. A negative test will result in a dark control line with a blank test line. In a positive test, reporter moieties have been trans-cleaved by the cascade assay, thereby separating the biotin terminus from the FAM terminus. For cleaved reporter moieties, nanoparticles are captured at the test line due to anti-FAM antibodies. This positive test results in a dark test line in addition to a dark control line.

The components of the cascade assay may be provided in various kits for testing at, e.g., point of care facilities, in the field, pandemic testing sites, and the like. In one aspect, the kit for detecting a target nucleic acid of interest in a sample includes: first ribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes (RNP2s), blocked nucleic acid molecules, and reporter moieties. The first complex (RNP1) comprises a first nucleic acid-guided nuclease and a first gRNA, where the first gRNA includes a sequence complementary to the target nucleic acid(s) of interest. Binding of the first complex (RNP1) to the target nucleic acid(s) of interest activates trans-cleavage activity of the first nucleic acid-guided nuclease. The second complex (RNP2) comprises a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest. The blocked nucleic acid molecule comprises a sequence complementary to the second gRNA, where trans-cleavage of the blocked nucleic acid molecule results in an unblocked nucleic acid molecule and the unblocked nucleic acid molecule can bind to the second complex (RNP2), thereby activating the trans-cleavage activity of the second nucleic acid-guided nuclease. Activating trans-cleavage activity in RNP2 results in an exponential increase in unblocked nucleic acid molecules and in active reporter moieties, where reporter moieties are nucleic acid molecules and/or are operably linked to the blocked nucleic acid molecules and produce a detectable signal upon cleavage by RNP2.

In a second aspect, the kit for detecting a target nucleic acid molecule in sample includes: first ribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes (RNP2s), template molecules, blocked primer molecules, a polymerase, NTPs, and reporter moieties. The first ribonucleoprotein complex (RNP1) comprises a first nucleic acid-guided nuclease and a first gRNA, where the first gRNA includes a sequence complementary to the target nucleic acid of interest and where binding of RNP1 to the target nucleic acid(s) of interest activates trans-cleavage activity of the first nucleic acid-guided nuclease. The second complex (RNP2) comprises a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest. The template molecules comprise a primer binding domain (PBD) sequence as well as a sequence corresponding to a spacer sequence of the second gRNA. The blocked primer molecules comprise a sequence that is complementary to the PBD on the template nucleic acid molecule and a blocking moiety.

Upon binding to the target nucleic acid of interest, RNP1 becomes active triggering trans-cleavage activity that cuts at least one of the blocked primer molecules to produce at least one unblocked primer molecule. The unblocked primer molecule hybridizes to the PBD of one of the template nucleic acid molecules, is trimmed of excess nucleotides by the 3′-to-5′ exonuclease activity of the polymerase and is then extended by the polymerase and NTPs to form a synthesized activating molecule with a sequence that is complementary to the second gRNA of RNP2 (i.e., the synthesized activating molecule is the target strand). Upon activating RNP2, additional trans-cleavage activity is initiated, cleaving at least one additional blocked primer molecule. Continued cleavage of blocked primer molecules and subsequent activation of more RNP2s proceeds at an exponential rate. A signal is generated upon cleavage of a reporter molecule by active RNP2 complexes; therefore, a change in signal production indicates the presence of the target nucleic acid molecule.

Any of the kits described herein may further include a sample collection device, e.g., a syringe, lancet, nasal swab, or buccal swab for collecting a biological sample from a subject, and/or a sample preparation reagent, e.g., a lysis reagent. Each component of the kit may be in separate container or two or more components may be in the same container. The kit may further include a lateral flow device used for contacting the biological sample with the reaction mixture, where a signal is generated to indicate the presence or absence of the target nucleic acid molecule of interest. In addition, the kit may further include instructions for use and other information.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.

Example I: Preparation of Nucleic Acids of Interest

Mechanical lysis: Nucleic acids of interest may be isolated by various methods depending on the cell type and source (e.g., tissue, blood, saliva, environmental sample, etc.). Mechanical lysis is a widely used cell lysis method and may be used to extract nucleic acids from bacterial, yeast, plant and mammalian cells. Cells are disrupted by agitating a cell suspension with “beads” at high speeds (beads for disrupting various types of cells can be sourced from, e.g., OPS Diagnostics (Lebanon NJ, US) and MP Biomedicals (Irvine, CA, USA)). Mechanical lysis via beads begins with harvesting cells in a tissue or liquid, where the cells are first centrifuged and pelleted. The supernatant is removed and replaced with a buffer containing detergents as well as lysozyme and protease. The cell suspension is mixed to promote breakdown of the proteins in the cells and the cell suspension then is combined with small beads (e.g., glass, steel, or ceramic beads) that are mixed (e.g., vortexed) with the cell suspension at high speeds. The beads collide with the cells, breaking open the cell membrane with shear forces. After “bead beating”, the cell suspension is centrifuged to pellet the cellular debris and beads, and the supernatant may be purified via a nucleic acid binding column (such as the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit from ThermoFisher (Waltham, MA, USA) and others from Qiagen (Hilden, Germany), TakaraBio (San Jose, CA, USA), and Biocomma (Shenzen, China)) to collect the nucleic acids (see the discussion of solid phase extraction below).

Solid phase extraction (SPE): Another method for capturing nucleic acids is through solid phase extraction. SPE involves a liquid and stationary phase, which selectively separates the target analyte (here, nucleic acids) from the liquid in which the cells are suspended based on specific hydrophobic, polar, and/or ionic properties of the target analyte in the liquid and the stationary solid matrix. Silica binding columns and their derivatives are the most commonly used SPE techniques, having a high binding affinity for DNA under alkaline conditions and increased salt concentration; thus, a highly alkaline and concentrated salt buffer is used. The nucleic acid sample is centrifuged through a column with a highly porous and high surface area silica matrix, where binding occurs via the affinity between negatively charged nucleic acids and positively charged silica material. The nucleic acids bind to the silica matrices, while the other cell components and chemicals pass through the matrix without binding. One or more wash steps typically are performed after the initial sample binding (i.e., the nucleic acids to the matrix), to further purify the bound nucleic acids, removing excess chemicals and cellular components non-specifically bound to the silica matrix. Alternative versions of SPE include reverse SPE and ion exchange SPE, and use of glass particles, cellulose matrices, and magnetic beads.

Thermal lysis: Thermal lysis involves heating a sample of mammalian cells, virions, or bacterial cells at high temperatures thereby damaging the cellular membranes by denaturizing the membrane proteins. Denaturizing the membrane proteins results in the release of intracellular DNA. Cells are generally heated above 90° C., however time and temperature may vary depending on sample volume and sample type. Once lysed, typically one or more downstream methods, such as use of nucleic acid binding columns for solid phase extraction as described above, are required to further purify the nucleic acids.

Physical lysis: Common physical lysis methods include sonication and osmotic shock. Sonication involves creating and rupturing of cavities or bubbles to release shockwaves, thereby disintegrating the cellular membranes of the cells. In the sonication process, cells are added into lysis buffer, often containing phenylmethylsulfonyl fluoride, to inhibit proteases. The cell samples are then placed in a water bath and a sonication wand is placed directly into the sample solution. Sonication typically occurs between 20-50 kHz, causing cavities to be formed throughout the solution as a result of the ultrasonic vibrations; subsequent reduction of pressure then causes the collapse of the cavity or bubble resulting in a large amount of mechanical energy being released in the form of a shockwave that propagates through the solution and disintegrates the cellular membrane. The duration of the sonication pulses and number of pulses performed varies depending on cell type and the downstream application. After sonication, the cell suspension typically is centrifuged to pellet the cellular debris and the supernatant containing the nucleic acids may be further purified by solid phase extraction as described above.

Another form of physical lysis is osmotic shock, which is most typically used with mammalian cells. Osmotic shock involves placing cells in DI/distilled water with no salt added. Because the salt concentration is lower in the solution than in the cells, water is forced into the cell causing the cell to burst, thereby rupturing the cellular membrane. The sample is typically purified and extracted by techniques such as e.g., solid phase extraction or other techniques known to those of skill in the art.

Chemical lysis: Chemical lysis involves rupturing cellular and nuclear membranes by disrupting the hydrophobic-hydrophilic interactions in the membrane bilayers via detergents. Salts and buffers (such as, e.g., Tris-HCl pH8) are used to stabilize pH during extraction, and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)) and inhibitors (e.g., Proteinase K) are also added to preserve the integrity of the nucleic acids and protect against degradation. Often, chemical lysis is used with enzymatic disruption methods (see below) for lysing bacterial cell walls. In addition, detergents are used to lyse and break down cellular membranes by solubilizing the lipids and membrane proteins on the surface of cells. The contents of the cells include, in addition to the desired nucleic acids, inner cellular proteins and cellular debris. Enzymes and other inhibitors are added after lysis to inactivate nucleases that may degrade the nucleic acids. Proteinase K is commonly added after lysis, destroying DNase and RNase enzymes capable of degrading the nucleic acids. After treatment with enzymes, the sample is centrifuged, pelleting cellular debris, while the nucleic acids remain in the solution. The nucleic acids may be further purified as described above.

Another form of chemical lysis is the widely used procedure of phenol-chloroform extraction. Phenol-chloroform extraction involves the ability for nucleic acids to remain soluble in an aqueous solution in an acidic environment, while the proteins and cellular debris can be pelleted down via centrifugation. Phenol and chloroform ensure a clear separation of the aqueous and organic (debris) phases. For DNA, a pH of 7-8 is used, and for RNA, a more acidic pH of 4.5 is used.

Enzymatic lysis: Enzymatic disruption methods are commonly combined with other lysis methods such as those described above to disrupt cellular walls (bacteria and plants) and membranes. Enzymes such as lysozyme, lysostaphin, zymolase, and protease are often used in combination with other techniques such as physical and chemical lysis. For example, one can use cellulase to disrupt plant cell walls, lysosomes to disrupt bacterial cell walls and zymolase to disrupt yeast cell walls.

Example II: RNP Formation

For RNP complex formation, 250 nM of LbCas12a nuclease protein was incubated with 375 nM of a target specific gRNA in 1× Buffer (10 mM Tris-HCl, 100 μg/mL BSA) with 2-15 mM MgCl₂ at 25° C. for 20 minutes. The total reaction volume was 2 μL. Other ratios of LbCas12a nuclease to gRNAs were tested, including 1:1, 1:2 and 1:5. The incubation temperature ranged from 16° C.-37° C., and the incubation time ranged from 10 minutes to 4 hours.

Example III: Blocked Nucleic Acid Molecule Formation

Ramp cooling: For formation of the secondary structure of blocked nucleic acid molecules, 2.5 μM of a blocked nucleic acid molecule (any of Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to 37° C. at 0.015° C./second to form the desired secondary structure.

Snap cooling: For formation of the secondary structure of blocked nucleic acid molecules, 2.5 μM of a blocked nucleic acid molecule (any of Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by removing the heat source to form the desired secondary structure.

Snap cooling on ice: For formation of the secondary structure of blocked nucleic acid molecules, 2.5 μM of a blocked nucleic acid molecule (any of Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by placing the reaction tube on ice to form the desired secondary structure.

Example IV: Reporter Moiety Formation

The reporter moieties used in the reactions herein were single-stranded DNA oligonucleotides 5-9 bases in length (e.g., with sequences of TTATT, TTTATTT, ATTAT, ATTTATTTA, AAAAA, or AAAAAAAAA) with a fluorophore and a quencher attached on the 5′ and 3′ ends, respectively. In one example using a Cas12a cascade, the fluorophore was FAM-6 and the quencher was IOWA BLACK® (Integrated DNA Technologies, Coralville, IA). In another example using a Cas13 cascade, the reporter moieties were single-stranded RNA oligonucleotides 5-10 bases in length (e.g., r(U)n, r(UUAUU)n, r(A)n).

Example V: Cascade Assay

Format I (final reaction mix components added at the same time): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the Methicillin resistant Staphylococcus aureus (MRSA) DNA according to the RNP complex formation protocol described in Example II (for this sequence, see Example VI). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, MA) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. Thereafter, the final reaction was carried out in 1×Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, MA) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, 15 nM LbCas12a: 22.5 nM gRNA RNP1, 20 nM LbCas12a: 35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of Formula I-IV) in a total volume of 9 μL. 1 μL of MRSA DNA target (with samples having as low as three copies and as many as 30,000 copies—see FIGS. 6-14 ) was added to make a final volume of 10 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.

Format II (RNP1 and MRSA target pre-incubated before addition to final reaction mix): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the MRSA DNA according to RNP formation protocol described in Example II (for this sequence, see Example VI). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, MA) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1 μL of MRSA DNA target and incubated at 16° C.-37° C. for up to 10 minutes to activate RNP1. The final reaction was carried out in 1× Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, MA) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, the pre-incubated and activated RNP1, 20 nM LbCas12a: 35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of Formula I-IV) in a total volume of 9 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.

Format III (RNP1 and MRSA target pre-incubated before addition to final reaction mix and blocked nucleic acid molecule added to final reaction mix last): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the MRSA DNA according to the RNP complex formation protocol described in Example II (for this sequence, see Example VI). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, MA) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1 μL of MRSA DNA target and incubated at 16° C.-37° C. for up to 10 minutes to activate RNP1. The final reaction was carried out in 1× Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, MA) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, the pre-incubated and activated RNP1, and 20 nM LbCas12a: 35 nM gRNA RNP2 in a total volume of 9 μL. Once the reaction mix was made, 1 μL (50 nM) blocked nucleic acid molecule (any one of Formula I-IV) was added for a total volume of 10 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.

Example VI: Detection of MRSA and Test Reaction Conditions

To detect the presence of Methicillin resistant Staphylococcus aureus (MRSA) and determine the sensitivity of detection with the cascade assay, titration experiments with a MRSA DNA target nucleic acid of interest were performed. The MRSA DNA sequence (NCBI Reference Sequence NC: 007793.1) is as follows.

SEQ ID NO: 615: ATGAAAAAGATAAAAATTGTTCCACTTATTTTAATAGTTGTAGTTGTCGG GTTTGGTATATATTTTTATGCTTCAAAAGATAAAGAAATTAATAATACTA TTGATGCAATTGAAGATAAAAATTTCAAACAAGTTTATAAAGATAGCAGT TATATTTCTAAAAGCGATAATGGTGAAGTAGAAATGACTGAACGTCCGAT AAAAATATATAATAGTTTAGGCGTTAAAGATATAAACATTCAGGATCGTA AAATAAAAAAAGTATCTAAAAATAAAAAACGAGTAGATGCTCAATATAAA ATTAAAACAAACTACGGTAACATTGATCGCAACGTTCAATTTAATTTTGT TAAAGAAGATGGTATGTGGAAGTTAGATTGGGATCATAGCGTCATTATTC CAGGAATGCAGAAAGACCAAAGCATACATATTGAAAATTTAAAATCAGAA CGTGGTAAAATTTTAGACCGAAACAATGTGGAATTGGCCAATACAGGAAC AGCATATGAGATAGGCATCGTTCCAAAGAATGTATCTAAAAAAGATTATA AAGCAATCGCTAAAGAACTAAGTATTTCTGAAGACTATATCAAACAACAA ATGGATCAAAATTGGGTACAAGATGATACCTTCGTTCCACTTAAAACCGT TAAAAAAATGGATGAATATTTAAGTGATTTCGCAAAAAAATTTCATCTTA CAACTAATGAAACAGAAAGTCGTAACTATCCTCTAGGAAAAGCGACTTCA CATCTATTAGGTTATGTTGGTCCCATTAACTCTGAAGAATTAAAACAAAA AGAATATAAAGGCTATAAAGATGATGCAGTTATTGGTAAAAAGGGACTCG AAAAACTTTACGATAAAAAGCTCCAACATGAAGATGGCTATCGTGTCACA ATCGTTGACGATAATAGCAATACAATCGCACATACATTAATAGAGAAAAA GAAAAAAGATGGCAAAGATATTCAACTAACTATTGATGCTAAAGTTCAAA AGAGTATTTATAACAACATGAAAAATGATTATGGCTCAGGTACTGCTATC CACCCTCAAACAGGTGAATTATTAGCACTTGTAAGCACACCTTCATATGA CGTCTATCCATTTATGTATGGCATGAGTAACGAAGAATATAATAAATTAA CCGAAGATAAAAAAGAACCTCTGCTCAACAAGTTCCAGATTACAACTTCA CCAGGTTCAACTCAAAAAATATTAACAGCAATGATTGGGTTAAATAACAA AACATTAGACGATAAAACAAGTTATAAAATCGATGGTAAAGGTTGGCAAA AAGATAAATCTTGGGGTGGTTACAACGTTACAAGATATGAAGTGGTAAAT GGTAATATCGACTTAAAACAAGCAATAGAATCATCAGATAACATTTTCTT TGCTAGAGTAGCACTCGAATTAGGCAGTAAGAAATTTGAAAAAGGCATGA AAAAACTAGGTGTTGGTGAAGATATACCAAGTGATTATCCATTTTATAAT GCTCAAATTTCAAACAAAAATTTAGATAATGAAATATTATTAGCTGATTC AGGTTACGGACAAGGTGAAATACTGATTAACCCAGTACAGATCCTTTCAA TCTATAGCGCATTAGAAAATAATGGCAATATTAACGCACCTCACTTATTA AAAGACACGAAAAACAAAGTTTGGAAGAAAAATATTATTTCCAAAGAAAA TATCAATCTATTAACTGATGGTATGCAACAAGTCGTAAATAAAACACATA AAGAAGATATTTATAGATCTTATGCAAACTTAATTGGCAAATCCGGTACT GCAGAACTCAAAATGAAACAAGGAGAAACTGGCAGACAAATTGGGTGGTT TATATCATATGATAAAGATAATCCAAACATGATGATGGCTATTAATGTTA AAGATGTACAAGATAAAGGAATGGCTAGCTACAATGCCAAAATCTCAGGT AAAGTGTATGATGAGCTATATGAGAACGGTAATAAAAAATACGATATAGA TGAATAA

Briefly, a RNP1 was preassembled with a gRNA sequence designed to target MRSA DNA. Specifically, RNP1 was designed to target a 20 bp region of the mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 616). An RNP2 was preassembled with a gRNA sequence designed to target the unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) blocked nucleic acid molecule U29 (FIG. 10A). The reaction mix contained the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl.

FIG. 10A shows the structure and segment parameters of molecule U29. Note molecule U29 has a secondary structure free energy value of −5.84 kcal/mol and relatively short self-hybridizing, double-stranded regions of 5 bases and 6 bases. FIGS. 10B-10H show the results achieved for detection of 3E4 copies, 30 copies, 3 copies and 0 copies of the mecA gene of MRSA (n=3) at 25° C. with varying concentrations of blocked nucleic acid, RNP2 and reporter moiety. FIG. 10B shows the results achieved when 100 nM blocked nucleic acid molecules, 10 nM RNP2s and 500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 10:1. Note first that with 3E4 copies, nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 28.06 at 0 minutes, a signal-to-noise ratio of 24.23 at 5 minutes, and a signal-to-noise ratio of 21.01 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 12.45 at 0 minutes, 14.07 at 5 minutes and 16.16 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.79 at 0 minutes, 1.64 at 5 minutes and is 2.04 at 10 minutes. Note the measured fluorescence at 0 copies increases only slightly over the 10- and 30-minutes intervals, resulting in a flat negative. A flat negative (the results obtained over the time period for 0 copies) demonstrates that there is very little non-specific or undesired signal generation in the system. Note that the negative when the ratio of blocked nucleic acid molecules to RNP2s is 10:1 is flatter than those in FIGS. 10C through 10H.

FIG. 10C shows the results achieved when 50 nM blocked nucleic acid molecules, 10 nM RNP2s and 500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. Note first that with 3E4 copies, again nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 12.85, a signal-to-noise ratio of 10.51 at 5 minutes, and a signal-to-noise ratio of 8.18 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.85 at 0 minutes, 6.44 at 5 minutes and 6.48 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.54 at 0 minutes, 1.61 at 5 minutes and is 1.71 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2.

FIG. 10D shows the results achieved when 50 nM blocked nucleic acid molecules, 10 nM RNP2s and 2500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 34.92, a signal-to-noise ratio of 30.62 at 5 minutes, and a signal-to-noise ratio of 25.81 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 7.97 at 0 minutes, 1.73 at 5 minutes and 10.50 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.65 at 0 minutes, 1.73 at 5 minutes and is 1.82 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s, but likely due to the 5×increase in the concentration of reporter moieties; however, note also that a higher concentration of reporter moieties allows for a higher signal-to-noise ratio for 3E4 and 30 copies of MRSA target.

FIG. 10E shows the results achieved when 100 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and 4 mM NaCl. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1 but double the concentration of both of these molecules than that shown in FIGS. 10C and 10D. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 11.89, a signal-to-noise ratio of 8.97 at 5 minutes, and a signal-to-noise ratio of 6.53 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.46 at 0 minutes, 5.85 at 5 minutes and 5.43 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.58 at 0 minutes, 1.65 at 5 minutes and is 1.80 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown in FIG. 10B. Note also that the ratio of blocked nucleic acid molecules to RNP2s (5:1) appears to be more important than the ultimate concentration (100 nM/20 nM) by comparison to FIG. 10D where the ratio of blocked nucleic acid molecules to RNP2s was also 5:1 however the concentration of blocked nucleic acid molecules was 50 nM and the concentration of RNP2 was 10 nM.

FIG. 10F shows the results achieved when 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and using a concentration of 4 mM NaCl. In this experiment the ratio of blocked nucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 25.85, a signal-to-noise ratio of 21.36 at 5 minutes, and a signal-to-noise ratio of 16.24 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.28 at 0 minutes, 6.19 at 5 minutes and 7.02 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is very low at 0 minutes, 1.53 at 5 minutes and is 1.73 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio for all concentrations was reduced at the 2.5:1 ratio of blocked nucleic acid molecules to RNP2s.

FIG. 10G shows the results achieved when 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and using a concentration of 10 mM NaCl. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 12.75, a signal-to-noise ratio of 7.78 at 5 minutes, and a signal-to-noise ratio of 3.66 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 6.09 at 0 minutes, 6.23 at 5 minutes and 3.58 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is very low at 0 minutes, 1.40 at 5 minutes and is 1.62 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio for all concentrations was reduced substantially at the 2.5:1 ratio of blocked nucleic acid molecules to RNP2s and that the NaCl concentration at 10 mM vs. 4 mM (FIG. 10F) did not make much of a difference.

FIG. 10H shows the results achieved when 100 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and using a concentration of 10 mM NaCl. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 77.38, a signal-to-noise ratio of 74.18 at 5 minutes, and a signal-to-noise ratio of 67.90 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.94 at 0 minutes, 7.45 at 5 minutes and 9.73 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.66 at 0 minutes, 2.13 at 5 minutes and is 2.38 at 10 minutes. Note the measured fluorescence at 0 copies increases slightly, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio for all concentrations was increased substantially at the 5:1 ratio of blocked nucleic acid molecules to RNP2s as compared to the 2.5:1 ration of blocked nucleic acid molecules to RNP2s. In summary, the results shown in FIGS. 10B-10H indicate that a 5:1 ratio of blocked nucleic acid molecules to RNP2s or greater leads to higher signal-to-noise ratios for all concentrations of MRSA target.

Example VII: Homology Modeling and Mutation Structure Analysis

The variant nucleic acid-guided nucleases presented herein were developed in the following manner: For protein engineering and amino acid substitution model predictions, a first Protein Data Bank (pdb) file with the amino acid sequence and structure information for the RNP comprising the base nucleic acid-guided nuclease to be mutated, the gRNA and a bound dsDNA target nucleic acid was obtained. (For structural information for RNPs comprising AsCas12s and LbCas12a, see, e.g., Yamano, et al., Molecular Cell, 67:633-45 (2017).) Desired and/or random amino acid substitutions were then “made” to the base nucleic acid-guided nuclease (LbCas12a), the resulting structural change to the base nucleic acid-guided nuclease due to each amino acid substitution was used to generate updated files for the resulting RNPs comprising each of the variant nucleic acid-guided nucleases using SWISS-MODEL and the original pdf file as a reference template. SWISS-MODEL worked well in the present case as the amino acid sequences of wildtype LbCas12a was known, as were the planned amino acid substitutions. The output of the updated files for each variant nucleic acid-guided nuclease included a root mean square deviation (RMSD) value for the structural changes compared to the RNP complex for wt LbCas12a in Angstrom units (i.e., a measurement of the difference between the backbones of wt LbCas12a and the variant nucleic acid-guided nuclease) and the updated pdb files of the variant nucleic acid-guided nucleases are further assessed at the point of mutations for changes in the hydrogen bonds compared to the reference original pdb file of the nuclease.

After SWISS modeling, an independent step for calculating free energy was performed using, e.g., a Flex ddG module based on the program Rosetta CM to extract locally destabilizing mutations. This was used as a proxy for amino acid interference with PAM regions of the DNA to assess the probability of unwinding of the target nucleic acid. (See, e.g., Shanthirabalan, et al., Proteins: Structure, Function, and Bioinformatics 86(8):853-867 (2018); and Barlow, et al., J. Physical Chemistry B, 122(21):5389-99 (2018).)

Generally, the results of the SWISS-Model and Rosetta analysis indicated that stable enzyme function related to the PAM domain would require a global RMSD value range from 0.1 to 2.1 angstroms, and the following ΔΔG Flex Values: for stabilizing mutations ΔΔG≤−1.0 kcal/mol; for neutral mutations: −1.0 kcal/mol<ΔΔG<1.0 kcal/mol; and for destabilizing mutations: ΔΔG≥1.0 kcal/mol. Sixteen single mutations were identified that, singly or in combination, met the calculated criteria. Structural modeling for mutations at four exemplary amino acid residues are described below.

FIG. 6A shows the result of protein structure prediction using Rosetta and SWISS modeling of wildtype LbCas12a (Lachnospriaceae bacterium Cas12a). Protein structure prediction using Rossetta and SWISS modeling of exemplary variants of wildtype LbCas12a are shown below.

Mutation 1, G532A: The structure of an RNP comprising the G532A variant nucleic acid-guided nuclease is shown in FIG. 11A. Modeling indicated the following changes to the wildtype LbCas12a structure with the G532A substitution (seen in FIG. 11A as a red residue): loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 595; no addition or loss of a hydrogen bond at amino acid residue 532. Per simulations, mutation G532A is a structurally stabilizing mutation. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 17.

TABLE 17 Mutation 1: G532A Global RMSD: 0.976 PI RMSD: 0.361 REC1 RMSD: 0.289 (235 to 235 atoms) WED RMSD: 0.306 (198 to 198 atoms) ΔΔG Flex Value: −1.13 PI = PAM-interacting domain of the G532A variant REC1 = REC1 domain of the G532A variant WED = WED domain of the G532A variant

Mutation 2, K538A: The structure of an RNP comprising the K538A variant nucleic acid-guided nuclease is shown at left in FIG. 11B. Modeling indicated the following changes to the wildtype LbCas12a structure with the K538A substitution (seen in FIG. 11B as a pink residue): loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 538; loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 595. Per simulations, mutation K538A is a structurally stabilizing mutation. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 18.

TABLE 18 Mutation 2: K538A Global RMSD: 0.990 PI RMSD: 0.376 REC1 RMSD: 0.305 (236 to 236 atoms) WED RMSD: 0.324 (194 to 194 atoms) ΔΔG Flex Value: 0.06 PI = PAM-interacting domain of the K538A variant REC1 = REC1 domain of the K538A variant WED = WED domain of the K538A variant

Mutation 3, Y542A: The structure of an RNP comprising the Y542A variant nucleic acid-guided nuclease is shown in FIG. 11C. Modeling indicated the following changes to the wildtype LbCas12a structure with the Y542A substitution (seen in FIG. 11C as a blue residue): loss of two hydrogen bonds with TS-PAM (target strand PAM) at amino acid residue 542; loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 538; loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 595. Per simulations, mutation Y542A is a structurally stabilizing mutation. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 19.

TABLE 19 Mutation 3: Y542A Global RMSD: 0.989 PI RMSD: 0.377 REC1 RMSD: 0.306 (237 to 237 atoms) WED RMSD: 0.338 (199 to 199 atoms) ΔΔG Flex Value: −2.06 PI = PAM-interacting domain of the Y542A variant REC1 = REC1 domain of the Y542A variant WED = WED domain of the Y542A variant

Mutation 4, K595A: The structure of an RNP comprising the K595A variant nucleic acid-guided nuclease is shown in FIG. 11D. Modeling indicated the following changes to the wildtype LbCas12a structure with the K595A substitution (seen in FIG. 11D as an orange residue): loss of two hydrogen bonds with TS-PAM (target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 538. Per simulations, mutation K595A is a structurally destabilizing mutation. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 20.

TABLE 20 Mutation 4: K595A Global RMSD: 0.976 PI RMSD: 0.361 REC1 RMSD: 0.289 (235 to 235 atoms) WED RMSD: 0.306 (198 to 198 atoms) ΔΔG Flex Value: 1.26 PI = PAM-interacting domain of the K595A variant REC1 = REC1 domain of the K595A variant WED = WED domain of the K595A variant

Mutation 5, Combination G532A, K538A, Y542A, and K595A: The structure of an RNP comprising the combination G532A/K538A/Y542A/K595A variant (“combination variant”) nucleic acid-guided nuclease is shown in FIG. 11E. Modeling indicated the following changes to the wildtype LbCas12a structure with the four substitutions: loss of five hydrogen bonds with TS-PAM (target strand PAM); loss of one hydrogen bond with NTS-PAM (non-target strand PAM). Per simulations, the combination variant is structurally stable. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 21.

TABLE 21 Mutation 5: G532A/K538A/Y542A/K595A Global RMSD: 0.966 PI RMSD: 0.351 REC1 RMSD: 0.261 (226 to 226 atoms) WED RMSD: 0.288 (200 to 200 atoms) ΔΔG Flex Value: −3.31 PI = PAM-interacting domain of the combination variant REC1 = REC1 domain of the combination variant WED = WED domain of the combination variant

Mutation 6, K595D: The structure of an RNP comprising the K595D variant nucleic acid-guided nuclease is shown in FIG. 11F. Modeling indicated the following changes to the wildtype LbCas12a structure at location 595 with this substitution: loss of two hydrogen bonds with TS-PAM (target strand PAM); loss of one hydrogen bond with NTS-PAM (non-target strand PAM); and gain of one hydrogen bond with NTS-PAM. Per simulations, the K595D variant is structurally unstable. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 22.

TABLE 22 Mutation 6: K595D Global RMSD: 1.001 PI RMSD: 0.367 (89 to 89 atoms) REC1 RMSD: 0.296 (235 to 235 atoms) WED RMSD: 0.320 (197 to 197 atoms) ΔΔG Flex Value: 2.04 PI = PAM-interacting domain of the combination variant REC1 = REC1 domain of the combination variant WED = WED domain of the combination variant

Mutation 7, K595E: The structure of an RNP comprising the K595E variant nucleic acid-guided nuclease is shown in FIG. 11G. Modeling indicated the following changes to the wildtype LbCas12a structure at location 595 with this substitution: loss of two hydrogen bonds with TS-PAM (target strand PAM); loss of one hydrogen bond with NTS; and no gain of hydrogen bonds. Per simulations, the K595E variant is structurally unstable. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 23.

TABLE 23 Mutation 6: K595E Global RMSD: 0.975 PI RMSD: 0.352 (89 to 89 atoms) REC1 RMSD: 0.264 (226 to 226 atoms) WED RMSD: 0.290 (198 to 198 atoms) ΔΔG Flex Value: 1.37 PI = PAM-interacting domain of the combination variant REC1 = REC1 domain of the combination variant WED = WED domain of the combination variant

Mutation 8, Combination K538A, Y542A, K595D: The structure of an RNP comprising the combination K538A/Y542A/K595D variant (“combination variant”) nucleic acid-guided nuclease is shown in FIG. 11H. Modeling indicated the following changes to the wildtype LbCas12a structure with the three substitutions: loss of two hydrogen bonds with TS (target strand) at position 595; loss of one hydrogen bond with NTS (non-target); combined loss of three hydrogen bonds at 532/242 positions; and gain of one hydrogen bond at 595. Per simulations, the combination variant is structurally destabilizing. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 24.

TABLE 24 Mutation 6: K538A, Y542A, K595D Global RMSD: 0.976 PI RMSD: 0.351 (89 to 89 atoms) REC1 RMSD: 0.261 (225 to 225 atoms) WED RMSD: 0.289 (198 to 198 atoms) ΔΔG Flex Value: 0.96 PI = PAM-interacting domain of the combination variant REC1 = REC1 domain of the combination variant WED = WED domain of the combination variant

Mutation 9, Combination K538A, Y542A, K595E: The structure of an RNP comprising the combination K538A/Y542A/K595E variant (“combination variant”) nucleic acid-guided nuclease is shown in FIG. 11I. Modeling indicated the following changes to the wildtype LbCas12a structure with the three substitutions: loss of two hydrogen bonds with TS (target strand) at position 595; loss of one hydrogen bond with NTS (non-target); combined loss of three hydrogen bonds at 532/242 positions. Per simulations, the combination variant is structurally stabilizing. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 25.

TABLE 25 Mutation 6: K538A, Y542A, K595E Global RMSD: 0.976 PI RMSD: 0.351 (89 to 89 atoms) REC1 RMSD: 0.261 (225 to 225 atoms) WED RMSD: 0.289 (198 to 198 atoms) ΔΔG Flex Value: −3.71 PI = PAM-interacting domain of the combination variant REC1 = REC1 domain of the combination variant WED = WED domain of the combination variant

In addition to amino acid substitutions, modifications, such as chemical modifications, can be made to amino acids identified by the structural and homology modeling described above. FIG. 6G illustrates an exemplary scheme for acetylating amino acid residue 595 in LbCas12a, a modification which prevents unwinding of dsDNA by blocking entry of a target nucleic acid into the RNP via steric hindrance. LbCas12a is combined with AcrVA5 and the reaction is incubated for 20 minutes at room temperature, resulting in LECas12a that has been acetylated at amino acid residue 595 (K595K^(AC)). (For a discussion and methods for disabling of Cas12a by ArVA5, see Dong, et al., Nature Structural and Molecular Bio., 26(4):308-14 (2019).) DsDNA is not a substrate for LbCas12a with a K595K^(AC) modification; however, ssDNA is a substrate for LbCas12a with a K595K^(AC) modification; thus, LbCas12a (K595K^(AC)) has the desired properties of the variant nucleic acid-guided nucleases described above. In addition to acetylation, phosphorylation and methylation of select amino acid residues may be employed.

Example VIII: Single-Strand Specificity of the Variant Nucleic Acid-Guided Nucleases

In vitro transcription/translation reactions were performed for variant LbaCas12a nucleases as noted in Table 26 using the nucleic acid sequences listed in Table 27:

TABLE 26 Template DNA for IVTT 250 ng gRNA concentration 100 nM DNA activator concentration 25 nM Probe concentration 500 nM Reaction volume 30 μL Reporter 5′-FAM-TTATTATT-IABkFQ-3′ Plate PCR plate 96-well, black Read temperature 25° C. Read duration 30 minutes Buffer NEB r2.1 New England Biolabs ®, Inc., Ipswich, MA) Na+ 50 mM Mg+2 10 mM

TABLE 27 Activator RunX fragment GCCTTCAGAAGAGGGTGCATTTTCAGGAGGAA (dsDNA + PAM) GCGATGGCTTCAGACAGCATATTTGAGTCATT (SEQ ID NO. 617) RunX fragment GCCTTCAGAAGAGGGTGCATGCACAGGAGGAA (dsDNA − PAM) GCGATGGCTTCAGACAGCATATTTGAGTCATT (SEQ ID NO. 618) Target region in AGGAGGAAGCGATGGCTTCAGA activator (SEQ ID NO. 619) gRNA LbaCas12a gRNA gUAAUUUCUACUAAGUGUAGAUAGGAGGAAG CGAUGGCUUCAGA (SEQ ID NO. 620)

The results are shown in FIGS. 12A-12G indicating the time for detection of dsDNA and ssDNA both with and without PAM sequences for purified wildtype LbaCas12a and three variants (K538A+K595A, K595A, and K538A+Y542+K595A, and unpurified engineered variants of LbaCas12a: K538D+Y542A+K595D, K595D, K538A+K595D, K538A+K595E, G532A+K538A+Y542A+K595A, K538A+Y542A+K595D, K538D+Y542A+K595A, K538D+Y542D+K595A, and K538E+Y542A+K595A. Note that all variant engineered nucleic acid-guided nucleases slowed down double-strand DNA detection to varying degrees, with the double and triple variants at positions K538, Y542 and K595 of wt LbaCas12a performing best in comparison to wt LbCas12a, while single-strand DNA detection remained high, both in single-strand DNA with a PAM and without a PAM. The following variants were particularly robust: K538D+Y542A+K595D, K538A+K595D, K538A+K595E, G532A+K538A+Y542A+K595A, K538D+Y542A+K595A, and K538D+Y542D+K595D.

FIGS. 13A and 13B show the sequence alignment of many different Cas12a nucleases and orthologs, including in some instances several alignments of the same Cas12a nuclease.

Example IX: Detection of Biomarker Alpha-Synuclein in CSF for Monitoring Progression of Parkinson's Disease

The biomarker α-synuclein, which is found in both aggregated and fibrillar form, has attracted attention as a biomarker of Parkinson's disease. Human α-synuclein is expressed in the brain in the neocortex, hippocampus, substantia nigra, thalamus and cerebellum. It is encoded by the SNCA gene that consists of six exons ranging in size from 42 to 1110 base pairs. The predominant form of α-synuclein is the full-length protein, but other shorter isoforms exist. C-terminal truncation of α-synuclein induces aggregation, suggesting that C-terminal modifications may be involved in Parkinson's pathology. Changes in the levels of α-synuclein have been reported in CSF of Parkinson' patients. The gradual spread of α-synuclein pathology leads to a high concentration of extracellular α-synuclein that can potentially damage healthy neurons. Here, the cascade assay is used to monitor the level of nucleic acids in cerebrospinal fluid (CSF) to monitor the levels of mRNA transcripts that when translated lead to a truncated α-synuclein protein.

A lumbar puncture is performed on an individual, withdrawing approximately 5 mL of cerebrospinal fluid (CSF) for testing. The CSF sample is then treated by phenol-chloroform extraction or oligo dT affinity resins via a commercial kit (see, e.g., the TurboCapture mRNA kit or RNeaxy Pure mRNA Bead Kit from Qiagen®). Briefly, two RNP1s are preassembled as described above in Example II with a first gRNA sequence designed to target the coding sequence of the mRNA transcribed from SNCA gene specific to the C-terminus region of α-synuclein to detect full-length α-synuclein and second gRNA sequence designed to target the coding sequence of the mRNA transcribed from SNCA gene specific to the N-terminus region of α-synuclein to detect all α-synuclein mRNAs. In addition to the gRNA, each RNP1 also comprises an LbCas13a nuclease (i.e., an RNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V. A readout is performed by comparing the level of N-terminus coding sequences detected (the level of total α-synuclein mRNA) versus the level of C-terminus coding sequences detected (the level of full-length α-synuclein mRNA).

Example X: Detection of Foot and Mouth Disease Virus from Nasal Swabs

Foot-and-mouth disease (FMD) is a severe and highly contagious viral disease. The FMD virus causes illness in cows, pigs, sheep, goats, deer, and other animals with divided hooves and is a worldwide concern as it can spread quickly and cause significant economic losses. FMD has serious impacts on the livestock trade a single detection of FMD will stop international trade completely for a period of time. Since the disease can spread widely and rapidly and has grave economic consequences, FMD is one of the animal diseases livestock owners dread most. FMD is caused by a virus, which survives in living tissue and in the breath, saliva, urine, and other excretions of infected animals. FMD can also survive in contaminated materials and the environment for several months under the right conditions.

A nasal swab is performed on a subject, such as a cow or pig, and the nucleic acids extracted using, e.g., the Monarch Total RNA Miniprep Kit (New England Biolabs®, Inc., Ipswich, MA). Briefly, an RNP1 is preassembled as described above in Example II with a gRNA sequence designed to a gene from the FMD virus (e.g., to a portion of NCBI Reference Sequence NC_039210.1) and an LbCas12a nuclease (i.e., a DNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V, and the readout is positive detection of FMD virus-specific DNA sequences.

Example XI: Detection of Sickle Cell Gene Sequences in Peripheral Blood

Sickle cell disease (SCD) is a group of inherited red blood cell disorders. In someone who has SCD, the hemoglobin is abnormal, which causes the red blood cells to become hard and sticky and look like a C-shaped farm tool called a “sickle.” The sickle cells die early, which causes a constant shortage of red blood cells; in addition, when the sickle-shaped blood cells travel through small blood vessels, they get stuck and clog the blood flow, causing pain and other serious complications such as infection and stroke.

One form of SCD is HbSS. Individuals who have this form of SCD inherit two genes, one from each parent, that code for hemoglobin “S.” Hemoglobin S is an abnormal form of hemoglobin that causes the red cells to become rigid and sickle shaped. This is commonly called sickle cell anemia and is usually the most severe form of the disease. Another form of SCD is HbSC. Individuals who have this form of SCD inherit a hemoglobin “S” gene from one parent and a gene for a different type of abnormal hemoglobin called “C” from the other parent. This is usually a milder form of SCD. A third form of SCD is HbS thalassemia. Individuals who have this form of SCD inherit a hemoglobin “S” gene from one parent and a gene for beta thalassemia, another type of hemoglobin abnormality, from the other parent. There are two types of beta thalassemia: “zero” (HbS beta0) and “plus” (HbS beta+). Those with HbS beta0-thalassemia usually have a severe form of SCD. People with HbS beta+-thalassemia tend to have a milder form of SCD.

A non-invasive prenatal test (NIPT) that uses only maternal cell-free DNA (cfDNA) from peripheral blood permits prenatal detection of sickle cell disease and beta thalassemia by screening without the need for paternal DNA. Such a screening enables patients and healthcare providers to make informed decisions about diagnostic testing and may expand gene therapy treatment options. A 10 mL peripheral blood draw is performed on a pregnant subject into a Streck tube. The blood is treated with lysis-binding buffer and proteinase K under denaturing conditions at 55° C. for 15 minutes in the presence of magnetic beads. Following the heating step, the mixture is incubated for 1 hour at room temperature with mixing every 10 minutes at 1200 rpm for 30 seconds on an Eppendorf themomixer. The beads are captured on a magnetic stand for 2 minutes, washed three times after which cfDNA is eluted by adding elution buffer and incubating for 5 minutes at 55° C. The cfDNA is further purified by diluting in 1:1 FTA (Fast Technology for Analysis) reagent, cat #WHAWB120204 (Sigma-Aldrich, USA), containing NaCl (sodium chloride); Tris; EDTA (ethylenediaminetetraacetic acid); TRITON-X-100 (t-Octylphenoxypolyethoxyethanol) and incubated for 10 minutes at room temperature. An additional bead purification step is performed using PCRClean DX beads, cat #C-1003-450 (ALINE Biosciences, USA). Alternatively, there are several kits available commercially that are designed to extract cfDNA including the BioChain® cfPure® Cell free DNA Extraction Kit (BioChain®, Newark, CA); the Monarch Genomic DNA Purification Kit and the Monarch HMW DNA Extraction Kit for Blood (New England Biolabs®, Inc., Ipswich, MA); and the cfDNA Purification Kit (Active Motif®, Carlsbad, CA).

For the cascade assay, three RNP1s are preassembled as described above in Example II with 1) gRNA sequence designed to detect the Hemoglobin S gene variant and an LbCas12a nuclease (i.e., an DNA-specific nuclease); 2) a gRNA sequence designed to detect the Hemoglobin C gene variant and an LbCas 12a nuclease (i.e., an DNA-specific nuclease); and 3) a gRNA sequence designed to detect the gene for beta thalassemia and an LbCas12a nuclease (i.e., an DNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V. The readout is detection of the Hemoglobin S gene variant, the detection of the Hemoglobin S variant and the Hemoglobin C variant, and the detection of the Hemoglobin S variant and the β-thalassemia gene.

Example XII: Detection of Donor-Derived Gene Sequences in Peripheral Blood of Transplant Patients

Costly and invasive tissue biopsies to detect allograft rejection after transplantation have numerous limitations; however, assays based on cell-free DNA (cfDNA) circulating fragments of DNA released from cells, tissues, and organs as they undergo natural cell death can improve the ability to detect rejection and implement earlier changes in management of the transplanted organ. Rejection, referring to injury of a donated organ caused by the recipient's immune system, often causes allograft dysfunction and even patient death. T-cell mediated acute cellular rejection occurs most often within the first 6 months post-transplant. Acute cellular rejection involves accumulation of CD4+ and CD8+ T-cells in the interstitial space of the allograft as the recipient's immune system recognizes antigens on the donated organ as foreign, initiating an immune cascade that ultimately leads to apoptosis of the targeted cells. As these cells die, genomic DNA is cleaved and fragments of donor derived-cfDNA are released to join the pool of recipient cfDNA in the blood. Using cfDNA as a biomarker for acute cellular rejection is advantageous since it is derived from the injured cells of the donated organ and therefore should represent a direct measure of cell death occurring in the allograft. Further, cfDNA maintains all of the genetic features of the original genomic DNA, allowing the genetic material released from the donated organ to be differentiated from the cfDNA derived from cells of the recipient that are undergoing natural apoptosis.

For organ transplants in which the donor is male and the recipient is female, this “sex mismatch” is leveraged to calculate donor derived-cfDNA levels from within the recipient's total cfDNA pool. Although this approach allows for confident diagnosis of rejection in the allograft, sex-mismatch between the donor and recipient is relatively infrequent and not universally applicable; thus, the presence of other genetic differences between the donor and recipient at a particular locus are leveraged to identify the origin of the circulating cfDNA. Ideally, the recipient would be homozygous for a single base (for example, AA) and at the same locus the donor would be homozygous for a different base (for example, GG). Given the genetic heterogeneity between individuals, hundreds to tens of thousands of potentially informative loci across the genome can be interrogated to distinguish donor derived-cfDNA from recipient cfDNA.

A 10 mL peripheral blood draw is performed on a transplantation subject into a Streck tube. The blood is treated with lysis-binding buffer and proteinase K under denaturing conditions at 55° C. for 15 minutes in the presence of magnetic beads. Following the heating step, the mixture is incubated for 1 hour at room temperature with mixing every 10 minutes at 1200 rpm for 30 seconds on an Eppendorf themomixer. The beads are captured on a magnetic stand for 2 minutes, washed three times after which cfDNA is eluted by adding elution buffer and incubating for 5 minutes at 55° C. The cfDNA is further purified by diluting in 1:1 FTA (Fast Technology for Analysis) reagent, cat #WHAWB120204 (Sigma-Aldrich, USA), containing NaCl (sodium chloride); Tris; EDTA (ethylenediaminetetraacetic acid); TRITON-X-100 (t-Octylphenoxypolyethoxyethanol) and incubated for 10 minutes at room temperature. An additional bead purification step is performed using PCRClean DX beads, cat #C-1003-450 (ALINE Biosciences, USA). Also, as stated above, there are several kits available commercially that are designed to extract cfDNA including the BioChain® cfPure® Cell free DNA Extraction Kit (BioChain®, Newark, CA); the Monarch Genomic DNA Purification Kit and the Monarch HMW DNA Extraction Kit for Blood (New England Biolabs®, Inc., Ipswich, MA); and the cfDNA Purification Kit (Active Motif®, Carlsbad, CA).

For the cascade assay, several to many different RNP1s are preassembled as described above in Example II with gRNA sequences designed to 1) query Y and/or X chromosome loci in sex mismatch transplantation cases; or 2) gRNA sequences designed to query various loci that are different in the genomic DNA of the recipient and the donor; along with an LbCas12a nuclease (i.e., an DNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V. The readout detects the level of donor-specific nucleic acid sequences.

Example XIII: Detection of Microbe Contamination in a Laboratory

DNA that is found in the environment is called “environmental DNA” or eDNA (e-DNA) for short, and it is formally defined as “genetic material obtained directly from environmental samples without any obvious signs of biological source material.” eDNA has been harnessed to detect rare or invasive species and pathogens in a broad range of environments. Samples are typically collected in the form of water, soil, sediment, or surface swabs. The DNA must then be extracted and purified to remove chemicals that may inhibit the cascade reaction. Surface wipe samples are commonly collected to assess microbe contamination in, e.g., a laboratory. The wipe test protocol consists of four distinct stages: removal of DNA from surfaces using absorbent wipes, extraction of DNA from the wipes into a buffer solution, purification of DNA, and analysis of the extract.

For sample collection, sterile 2×2 inch polyester-rayon non-woven wipes are used to wipe down an environmental surface, such as a laboratory bench. Each wipe is placed into a sterile 50 ml conical tube and 10 mL of PBST is transferred to each conical tube using a sterile serological pipette. The tubes are vortexed at the maximum speed for 20 minutes using a Vortex Genie 2. A 200 μL aliquot of the supernatant was processed using a nucleic acid purification kit (QIAmp DNA Blood Mini Kit, QIAGEN, Inc., Valencia, CA). The kit lyses the sample, stabilizes and binds DNA to a selective membrane, and elutes the DNA sample.

For the cascade assay, several to many different RNP1s are preassembled as described above in Example II with gRNA sequences designed to detect, e.g., Aspergillus acidus; Parafilaria bovicola; Babesia divergens; Escherichia coli; Pseudomonas aeruginosa; and Dengue virus; along with an LbCas12a nuclease (i.e., an DNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V. The readout is detection of a genomic sequence unique to a pathogen.

While certain embodiments have been described, these embodiments have been presented by way of example only and are not intended to limit the scope of the present disclosures. Indeed, the novel methods, apparatuses, modules, instruments and systems described herein can be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the methods, apparatuses, modules, instruments and systems described herein can be made without departing from the spirit of the present disclosures. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the present disclosures. 

We claim:
 1. A method for preventing unwinding of blocked nucleic acid molecules in the presence of an RNP comprising the steps of: providing blocked nucleic acid molecules; providing ribonucleoprotein complexes comprising a Cas12a nucleic acid-guided nuclease that exhibits both cis- and trans-cleavage activity upon activation and a gRNA that recognizes an unblocked nucleic acid molecule resulting from trans-cleavage of the blocked nucleic acid molecules; and engineering the nucleic acid-guided nuclease used in the ribonucleoprotein complex to result in a variant nucleic acid-guided nuclease where single stranded DNA is cleaved faster than double stranded DNA is cleaved.
 2. The method of claim 1, wherein the blocked nucleic acid molecules comprise a structure represented by any one of Formulas I-IV, wherein Formulas I-IV are in the 5′-to-3′ direction: (a)A-(B-L)_(J)-C-M-T-D  (Formula I); wherein A is 0-15 nucleotides in length; B is 4-12 nucleotides in length; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)_(J)-C and T-D are separate nucleic acid strands; T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; and D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; (b)D-T-T′-C-(L-B)_(J)-A  (Formula II); wherein D is 0-10 nucleotides in length; T-T′ is 17-135 nucleotides in length; T′ is 1-10 nucleotides in length and does not hybridize with T; C is 4-15 nucleotides in length and comprises at least 50% sequence complementarity to T; L is 3-25 nucleotides in length and does not hybridize with T; B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T; J is an integer between 1 and 10; A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D; (c)T-D-M-A-(B-L)_(J)-C  (Formula III); wherein T is 17-135 nucleotides in length; D is 0-10 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L)_(J)-C are separate nucleic acid strands; A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D; B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T; L is 3-25 nucleotides in length; J is an integer between 1 and 10; and C is 4-15 nucleotides in length; or (d)T-D-M-A-L_(p)-C  (Formula IV); wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or 17-25); D is 0-15 nucleotides in length; M is 1-25 nucleotides in length; A is 0-15 nucleotides in length and comprises a sequence complementary to D; and L is 3-25 nucleotides in length; p is 0 or 1; C is 4-15 nucleotides in length and comprises a sequence complementary to T.
 3. The method of claim 1, wherein the RNP comprises a variant nucleic acid-guided nuclease comprising at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecule and wherein the mutation is selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs.
 4. The method of claim 3, wherein there are at least two mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs.
 5. The method of claim 4, wherein there are at least three mutations to domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecule selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs. 